U.S. patent number 5,750,767 [Application Number 08/466,319] was granted by the patent office on 1998-05-12 for synthesis and use of amino acid fluorides as peptide coupling reagents.
This patent grant is currently assigned to Research Corporation Technologies, Inc.. Invention is credited to Louis A. Carpino, Ayman Ahmed El-Faham.
United States Patent |
5,750,767 |
Carpino , et al. |
May 12, 1998 |
**Please see images for:
( Certificate of Correction ) ** |
Synthesis and use of amino acid fluorides as peptide coupling
reagents
Abstract
The present invention is directed to the process of preparing a
peptide comprising reacting a first amino acid or peptide with an
amino acid fluoride of the formula: ##STR1## or the acid fluoride
salts thereof wherein BLK is an N-amino protecting group AA is an
amino acid residue and X is H or a protecting group useful, and the
first amino and peptide have a free amino group and a blocked
carboxy end.
Inventors: |
Carpino; Louis A. (Amherst,
MA), El-Faham; Ayman Ahmed (Amherst, MA) |
Assignee: |
Research Corporation Technologies,
Inc. (Tucson, AZ)
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Family
ID: |
23092202 |
Appl.
No.: |
08/466,319 |
Filed: |
June 6, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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284964 |
Aug 2, 1994 |
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426121 |
Oct 23, 1989 |
5360928 |
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Foreign Application Priority Data
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Oct 22, 1990 [WO] |
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PCT/US90/06061 |
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Current U.S.
Class: |
560/161; 530/333;
530/334; 530/335; 530/336; 530/337; 530/338; 544/334; 546/186;
548/300.1; 548/340.1; 548/343.1; 548/347.1; 548/495; 548/496;
548/497; 548/527; 548/530 |
Current CPC
Class: |
C07C
271/10 (20130101); C07C 271/34 (20130101); C07C
275/40 (20130101); C07C 275/70 (20130101); C07C
317/04 (20130101); C07C 317/10 (20130101); C07C
317/14 (20130101); C07C 317/18 (20130101); C07C
321/14 (20130101); C07C 321/20 (20130101); C07C
323/14 (20130101); C07D 207/16 (20130101); C07D
209/20 (20130101); C07D 233/68 (20130101); C07D
295/215 (20130101); C07K 1/088 (20130101); C07K
1/10 (20130101); Y02P 20/55 (20151101); C07C
2603/18 (20170501) |
Current International
Class: |
C07C
275/00 (20060101); C07C 275/40 (20060101); C07C
257/14 (); C07C 257/18 (); C07D 233/02 (); C07D
207/02 () |
Field of
Search: |
;530/333,334,335,336,337,338 ;544/334 ;546/186
;548/300.1,340.1,343.1,347.1,495,496,497,527,530 ;552/104
;560/29,30,160,161 ;562/553,845,849,851,852
;564/1,225,245,433,492,510 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Rajeswari, et al. "A New Synthesis of Amides From Acyl Fluorides
and N-silyamines", Tetrahedron Letters, 28, 43, pp. 5099-5102,
1987. .
Olah, et al., Synthetic Methods and Reactions; IV.sup.1
Fluorination of Carboxylic Acids With Cyanuric Fluoride,
Communications, 487-488, 1973. .
Picard, et al., Synthese de Dilactones Macroccliues Asistee Par Les
Organostanniques Application AUX Macrocycles Sourfres (Fulfures,
Sulfoxydes, Disulfures) Complexation Selective de L'ion CA.sup.2+,
Tetrahedron, 42, 13, 3503-3519, 1986. .
Carpino, et al. (Fluorenylmethyl)OXY)Carbonyl(FMOC) Amino Acid
Fluorides. Convenient New Peptide Coupling Reagents Applicable To
The FMOC/ Tert-Butyl Strategy For Solution And Solid-Phase
Syntheses, J. Am. Chem. Soc. 1990, 112, 9651-9652. .
Carter and Hinman, J. Biol. Chem., 1949, 178, 403, 409, 413. .
Jones, et al., J. Chem. Soc., 1979, 3203. .
Mobashery and Johnston, J. Org. Chem., 1985, 50, 2000. .
Schmidt, et al., Synthesis, 1988, 475, 476. .
Beyerman & Carpino, Peptides, "Use of RMOC Amino Acid Chlorides
. . . ". .
Beecham, JACS, vol. 79, 3257-3261 (1957). .
Mobashery, et al., "A New Approach to the Preparation of N-Carboxy
.alpha.-Amino Acid Anhydrides", J. Org. Chem., 50: 2200-2202
(1985). .
Beecham, "Tosyl-.alpha.-Amino Acids", II. The Use of Acid Chlorides
for Peptide Synthesis in the Presence of Aqueous Alkali, J. Am.
Chem. Soc., 79: 3262-3263 (1957). .
Wiley, et al., "Base-Catalyzed Decomposition of .alpha.-(Benzene-
and p-Toluene-Sulfonamido)-Phenylacetyl Chlorides", J. Am. Chem.
Soc., 74: 936-938 (1952). .
Wiley, et al., "Decarboxylation of
.alpha.-(Benzenesulfonamide)-Carboxylic Acids", J. Am. Chem. Soc.,
73: 4719-4720 (1951). .
Wiley, et al., Based-Catalyzed Decomposition of Substituted
.alpha.-(Benzene-Sulfonamide)-Carboxylic Acids and Their Acyl
Chlorides, J. Am. Chem. Soc., 76: 3496-3499 (1954). .
M. Bodanszky, Principles of Peptide Synthesis, 102-107, (1984).
.
Jakubke, et al., Amino Acids, Peptides and Proteins, 85, 88.
(1965). .
Beyermann, et al. J. Org. Chem., 1990, 55, 721-728. .
Chemical Abstracts 1984, 100, 103887. .
Chemical Abstracts 1985, 102:25040A. .
Carpino, et al., Journal of Organic Chemistry, 1986, 51, 3732-3734.
.
Carpino, et al. J. Amer. Chem. Soc., 112, 9651, (1990). .
Carpino, et al., J. Org. Chem., 56, 2611, (1991). .
Dourtaglou, et al., Synthesis, 572 (1984). .
Carpino, et al., J. Org. Chem., 56, 2635, (1991). .
Mukaiyama, Angew. Chem. I.E. Eng., 18, 707 (1979). .
Wenschuh, et al., Tetrahedron Lett., 3733, (1993). .
Coste, et al., Tetrahedron Lett., 1967, (1991). .
Coste, et al., Tetrahedron Lett., 669, (1990). .
Carpino, et al., J. Org. Chem., 57, 6371, (1992). .
Yamada, et al., Mem. Konan Univ. Sci. Ser., 32, 11, (1985). .
Carpino, et al., J. Amer. Chem. Soc., 115, 4397, (1993). .
Carpino, et al., J. Org. Chem., 55, 1673, (1990). .
Carpino, et al., J. Org. Chem., 55, 721, (1990)..
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Primary Examiner: O'Sullivan; Peter
Attorney, Agent or Firm: Scully, Scott, Murphy &
Presser
Government Interests
GOVERNMENT SPONSORSHIP
This work has been supported by a grant from the National
Institutes of Health, (Grant No. Gm-09706) and a grant from the
National Science Foundation (Grant No. CHE-8609176). The Government
has certain rights in the invention.
Parent Case Text
RELATED APPLICATIONS
This is a continuation of application Ser. No. 08/284,964, filed on
Aug. 2, 1994, which is a CIP application of U.S. Ser. No. 426,121,
filed Oct. 23, 1989, now U.S. Pat. No. 5,360,928.
Claims
What is claimed is:
1. A process for preparing a protected amino acid fluoride of the
formula: ##STR48## or the hydrogen fluoride salts thereof wherein
BLK is an N-amino protecting group;
AA is an amino acid residue; and
X is a--or a protecting group,
comprising reacting a protected amino acid with a
fluoroformamidinium salt of the formula ##STR49## wherein R.sub.15,
R.sub.16, R.sub.17 and R.sub.18 are independently lower alkyl,
aryl, aryl lower alkyl, cycloalkyl, cycloalkyl lower alkyl or
R.sub.15 and R.sub.16, taken together with the nitrogen atom to
which they are attached form a 5- or 6- membered ring containing a
nitrogen ring atom and 4 or 5 carbon ring atoms and up to a total
of 10 carbon atoms, or
R.sub.17 and R.sub.18, taken together with the nitrogen atom to
which they are attached form a 5- or 6- membered ring containing a
nitrogen ring atom and 4-5 ring carbon atoms and up to a total of
10 carbon atoms or
R.sub.16 and R.sub.17 and taken together with the nitrogen atoms to
which they are attached and the carbon atom attached to said
nitrogen atoms form a 5- or 6- membered ring containing 2 nitrogen
ring atoms and 3-4 ring carbon atoms and up to a total of 10 carbon
atoms and A is a counterion.
2. The process according to claim 1 wherein R.sub.15, R.sub.16,
R.sub.17 and R.sub.18 are independently lower alkyl or aryl or
R.sub.15 and R.sub.16 are taken together with the nitrogen atom to
which they are attached form a ring containing a nitrogen ring atom
or
R.sub.17 and R.sub.18 taken together with the nitrogen atom to
which they are attached form a ring containing a nitrogen ring atom
or
R.sub.16 and R.sub.17 taken together with the nitrogen atoms to
which they are attached and the carbon atom attached to said
nitrogen atoms form a 5- or 6-membered ring containing 2 ring
nitrogen atoms.
3. The process of claim 1 wherein R.sub.15, R.sub.16, and R.sub.17
are lower alkyl and R.sub.3 is lower alkyl or aryl.
4. The process of claim 1 wherein R.sub.15 and R.sub.16 are the
same.
5. The process of claim 1 wherein R.sub.15, R.sub.16 and R.sub.17
are the same.
6. The process of claim 1 wherein R.sub.15, R.sub.16, R.sub.17 and
R.sub.18 are the same.
7. The process of claim 1 wherein lower alkyl is alkyl containing
1-3 carbon atoms.
8. The process of claim 1 wherein lower alkyl is methyl and aryl is
phenyl.
9. The process of claim 1 wherein R.sub.15, R.sub.16 and R.sub.17
are all methyl and R.sub.18 is methyl or phenyl.
10. The process of claim 1 wherein R.sub.15 and R.sub.16 taken
together with the nitrogen atom to which they are attached form a
piperidine or pyrrolidine.
11. The process of claim 1 wherein R.sub.17 and R.sub.18 taken
together with the nitrogen atom to which they are attached form a
piperidine or pyrrolidine.
12. The process of claim 1 wherein R.sub.15 and R.sub.16 taken
together with the nitrogen atom to which they are attached and
R.sub.17 and R.sub.18 taken together with the nitrogen atom to
which they are attached form a piperidine or pyrrolidine.
13. The process of claim 1 wherein R.sub.16 and R.sub.17 taken
together with the nitrogen atoms to which they are attached and the
carbon atom attached to said nitrogen atoms form the
fluoroformamidinium salt which has the formula: ##STR50##
14. The process of claim 1 wherein the cationic portion of the
fluoroformamidinium salt is tetramethyl fluoroformamidinium,
trimethylphenyl fluoroformamidinium,
bis(tetramethylene)fluoroformamidinium or
1,3-dimethyl-2-fluoroimidizolium.
15. The process of claim 1 wherein AA is an .alpha.-amino acid.
16. The process of claim 1 wherein AA is glycine, alanine, leucine,
isoleucine, proline, hydroxyproline, phenylalanine, tyrosine,
methionine, norleucine, serine, threonine, cystine, cysteine,
tyrosine, aspartic acid, glutamic acid, asparagine, glutamine,
lysine, arginine, hydroxy lysine, ornithine, histidine or
tryptophan.
17. The process of claim 1 wherein BLK is CBz, Bspoc, Bsmoc, FMOC,
BOC, AIMOC, BIMOC, Dbd-TMOC, AOC, ADOC, Mch, Bpoc, Ddz, HCO, TFA,
TEOC, MOZ, Tac, Benz[e]indene-1-methoxy carbonyl,
Benz-[e]-indene-3-methoxycarbonyl, piperidine-oxycarbonyl,
phthaloyl, Azoc, Poc, Fac, Nps, Dts, formyl, acetyl or
trifluoroacetyl.
18. The process of claim 1 wherein BLK is Bspoc, Bsmoc, FMOC, BPoc,
BOC, CBZ, formyl or trifluoroacetyl.
Description
BACKGROUND OF THE DISCLOSURE
1. Field of the Invention
The present invention relates to novel amino acid fluorides and
protected amino acid fluorides and their use in synthetic
biochemistry, including peptide syntheses. More particularly, this
invention is directed to the N-protected amino acid fluorides, and
free amino acid fluorides and the hydrogen fluoride salts thereof,
the side chain of which may be unprotected or protected with a
blocking group and their use thereof in peptide synthesis.
2. Background of the Prior Art
As more and more polypeptides become of medicinal importance, there
is an increasing incentive to improve the methods by which they may
be synthesized. In recent years, peptides which have been found to
be of possible pharmacological importance include those active
against various diseases, such as cancers, diabetes, and plant
toxins, etc. Others have shown specific activity as growth
promoters or suppressants, antibiotics, insecticides,
contraceptives, anti-hypertensives, sleep-inducers,
anti-depressants, analgesics, etc. The list is long and varied.
Currently, syntheses of peptides in solution by classic or various
repetitive methods or on a solid support (Merrifield) are popular
techniques. Solution methods have the advantages of being easily
monitored and allowing purification of intermediates, if necessary,
at any stage. A major drawback is the relative slow pace of the
synthesis with each step being carried out manually.
The major advantage of the Merrifield Method is its easy automation
so that unattended, computer-controlled machine synthesis is
possible. Unfortunately, this method suffers from an inherent
deficiency due to the insoluble nature of the support on which the
synthesis proceeds. Unless each acylation step occurs with 100%
efficiency, mixtures will inevitably be built up on the polymer.
The longer the chain, the greater will be the contamination due to
undesired side reactions. Products produced in such reactions
remain to contaminate the desired product when at the end of the
cycle it is removed from the polymeric matrix. The properties of
these peptides will not differ sufficiently for peptides of greater
than about 30-40 residues to make efficient separation
feasible.
For very long segments (50 or more amino acids), therefore, current
methods are not satisfactory. Often mixtures are obtained of such
forbidding complexity that it may be difficult or impossible to
isolate the desired peptide.
The problems enumerated hereinabove could be eliminated if the
proper derivatives of the underlying amino acids and the proper
reaction conditions could be found.
For example, FMOC, (N.alpha.-(9-fluorenylmethyl)oxycarbonyl),
protected amino acid chlorides, which are described by Carpino, et
al. in J. Org. Chem. 51, 3732 (1986) have been used as acylating
agents for stepwise peptide syntheses for both solution and solid
phase techniques.
However, the amino acid chlorides have major drawbacks associated
therewith. First, the acid chlorides react with trace amounts of
water, such as moisture in the air, to give the corresponding amino
acid. Therefore, they are not so stable, and as such, they are not
a prime candidate for long term storage. Consequently, an objective
was to find an amino acid derivative which was stable to
moisture.
Moreover, another problem associated with amino acid chlorides is
that it has not been possible to date to synthesize amino acid
chlorides in which the protecting groups on the side chains of the
amino acids can be removed under extremely mild conditions. As one
skilled in the art is well aware, many of the amino acids have
functional groups on the side chains which can interfere with
peptide formation unless otherwise protected. In peptide synthesis,
only the mildest conditions should be used to remove these
protecting groups. For example, one of the easiest protecting
groups to remove from the side chains containing amino, hydroxyl or
carboxyl functions, such as lysine, tyrosine, threonine, serine,
aspartic acid, glutamic acid and the like, is t-butyl or t-butyl
containing moieties. For example, trifluoracetic acid can easily
remove the t-butyl group from a serine side chain; on the other
hand, a benzyl protecting group on the side chain can not be
removed by said treatment but instead requires a more potent acid
such as HF or trifluoromethanesulfonic acid. Therefore, the
conditions for removing the benzyl group from the side chain are
much harsher relative to the t-butyl groups. Furthermore, the mild
catalytic hydrogenolysis of benzyl groups is not generally
applicable to long chain peptides or resin attached peptides.
Although benzyl groups on the side chains of N-protected amino acid
chlorides, can be prepared, such as FMOC-cysteine-S-benzyl
chloride, FMOC-lysine-.epsilon. carbobenzoxy chloride,
FMOC-tyrosine-O-benzyl chloride, FMOC-serine-O-benzyl chloride and
FMOC aspartic acid .beta.-benzyl ester, these molecules suffer from
the disadvantages described hereinabove. Consequently, an
investigation was commenced to determine if t-butyl or "t-butyl
like" containing groups can be used to protect the side chain of
amino acid chlorides. Unfortunately, efforts in this area were
unsuccessful. None of the above compounds could be synthesized if
the t-butyl group was used in place of the benzyl substitution.
This was not unusual since it is well known that t-butyl-based
protecting groups are readily deblocked by hydrogen chloride which
is an inevitable by-product of acid chloride formation and/or long
term storage (hydrolysis by trace amounts of water). For example,
in the case of the FMOC-tyrosine derivative 1, the acid chloride
could be obtained, but after several days it was noted to lose the
t-butyl group slowly. ##STR2##
Furthermore, compound 1 as well as the analogous serine and
threonine derivatives could be obtained only as oils which could
not be crystallized and were therefore difficult, if not
impossible, to purify.
In the case of the FMOC aspartic acid derivative 4, treatment with
thionyl chloride gave only the aspartic acid anhydride 6,
presumably via the unstable acid chloride 5 which undergoes
intramolecular loss of t-butyl chloride. ##STR3##
Lysine derivative 7 could not be converted to an acid chloride
because of the marked sensitivity of the BOC function. ##STR4##
Similar problems arise in the cases of Arg, His, Asn, Gln, and Trp.
The net result of these problems is that only about one half of the
commonly occurring amino acids can be converted to stable amino
acid chlorides.
Therefore, a search was undertaken to find an amino acid candidate
for use in peptide synthesis which is inexpensive, stable to
moisture, and which shows great potential for long-term storage.
Moreover, it was hoped that a candidate could be found where the
protecting groups on the amino acid side chain could be removed
under milder conditions then those used to remove the benzyl group.
Preferably, it was hoped that a t-butyl containing group or a group
as easily removable as t-butyl could be placed on the side chain of
these amino acid candidates.
The present invention circumvents the difficulties experienced with
respect to the acid chlorides and accomplishes the goals described
hereinabove. The compounds of the present invention are effective
in coupling with amino acids or peptides to form new peptide bonds.
Moreover, the compounds of the present invention are more stable to
moisture then the acid chlorides and therefore can be used for long
term storage. Furthermore, t-butyl containing protecting groups and
other protecting groups can be placed on the side chains of these
amino acid compounds and removed under milder conditions than those
required for the removal of benzyl groups. Finally, the compounds
of the present invention are potent acylating agents in peptide
bond formation.
These compounds are, much to our surprise, the corresponding amino
acid fluorides.
SUMMARY OF THE INVENTION
The present invention is directed to an amino acid of the formula:
##STR5## or the hydrogen fluoride salts thereof wherein BLK is an
N-amino protecting group;
AA is an amino acid residue; and
X is --or a protecting group.
The present invention is also directed to a method for preparing a
peptide which comprises reacting the amino acid fluoride described
hereinabove with an amino acid or peptide having a free amino group
and removing the protecting groups therefrom.
In addition, the present invention is directed to a method for
preparing amino acid fluorides using a new fluorinating agent, a
fluoroformamidinium salt, of the formula: ##STR6## wherein
R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are independently lower
alkyl, aryl, aryl lower alkyl, cycloalkyl, cycloalkyl lower alkyl
or R.sub.15 and R.sub.16 taken together with the nitrogen atom to
which they are attached form a 5- or 6-membered ring or R.sub.17
and R.sub.18 taken together with the nitrogen atom to which they
are attached form a 5- or 6-membered ring or R.sub.15, R.sub.16,
R.sub.17 and R.sub.18 taken together with the nitrogen atom to
which they are attached form a 5- or 6-membered ring and A.sup.- is
a counter ion.
The present invention is also directed to use of the
fluoroformamidinium agent as a coupling agent for the assembly of
peptides.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "amino acid" refers to an organic acid
containing both a basic amino group (NH.sub.2) and an acidic
carboxyl group (COOH). Therefore, said molecule is amphoteric and
exists in aqueous solution as dipole ions. (See, "The Condensed
Chemical Dictionary", 10th ed. edited by Gessner G. Hawly, Van
Nostrand Reinhold Company, London, Eng. p.48 (1981)). The preferred
amino acids are the .alpha.-amino acids. They include but are not
limited to the 25 amino acids that have been established as protein
constituents. They must contain at least one carboxyl group and one
primary or secondary amino group on the amino acid molecule. They
include such proteinogenic amino acids as alanine, valine, leucine,
isoleucine, norleucine, proline, hydroxyproline, phenylalanine,
tryptophan, methionine, glycine, serine, threonine, cysteine,
cystine, tyrosine, asparagine, glutamine, aspartic acid, glutamic
acid, lysine, hydroxylysine, ornithine, arginine, histidine,
penicillamine and the like.
An "amino acid residue", as defined herein, is an amino acid minus
an amine hydrogen on the amino end of the molecule and the OH group
on the carboxy end of the molecule (i.e., it includes the acyl
group ##STR7## on the carboxy end of the molecule). Therefore,
unless designated to the contrary, the group "AA" signifies an
amino acid residue. For example, the amino acid residues of various
amino acids are represented below:
______________________________________ AA Symbol AA Residue
______________________________________ Gly ##STR8## Ala ##STR9##
Leu ##STR10## Ile ##STR11## Pro ##STR12## Phe ##STR13## Trp
##STR14## Met ##STR15## Ser ##STR16## Thr ##STR17## Cys ##STR18##
Tyr ##STR19## Asn ##STR20## Gln ##STR21## Asp ##STR22## Glu
##STR23## Lys ##STR24## Arg ##STR25## His ##STR26## Nor ##STR27##
______________________________________
Therefore, the symbol "AA-F" refers to an amino acid fluoride,
i.e., a compound having a fluoro group attached to the acyl group
##STR28## of the amino acid
When BLK is hydrogen, then the structure becomes the amino acid
fluoride of an amino acid having a free amino group. However, in
view of the synthesis of the amino acid fluoride described
hereinbelow, the free amino acid fluoride may be isolated as the
hydrogen fluoride salt thereof.
It will be apparent to one skilled in the art, shown by
exemplification in the table hereinabove that in the course of
protein synthesis, it may be necessary to protect certain side
chains of the amino acids to prevent unwanted side reactions. For
example, it may be necessary to protect the hydroxyl group on the
side chain of tyrosine, serine, or threonine in order to prevent
these groups from interfering with the desired reactions. This is a
common problem in peptide synthesis and many procedures are
available for protecting the functional groups on the side chains
of the amino acids. Such procedures for protecting various
functional groups are known to one skilled in the art and are
described in the treatise entitled "The PEPTIDES", Vol. 2, Edited
by E. Gross and J. Meienhoffer, Academic Press, N.Y., N.Y., pp.
166-251 (1980), and the book entitled "Reagents for Organic
Synthesis", by T. W. Green, John Wiley and Sons, New York, 1981,
the contents of both being incorporated herein by reference.
For example, when the functional side chain contains an hydroxy
group, such as threonine or serine, it can be protected by such
groups as methyl, methoxymethyl(MOM), 2-methoxyethoxymethyl(MEM),
tetrahydropyranyl, .beta.-trimethylsilylethyl,
4-methoxytetrahydropryanyl, l-ethoxyethyl, t-butyl,
p-methoxybenzyl, p-halobenzyl, o-nitrobenzyl, p-nitrobenzyl,
o-chlorobenzyl, adamantyl, diphenylmethyl, triphenylmethyl,
cyclohexyl, cyclopentyl, 1-benzyloxycarbonyl, tri-substituted
silyl, wherein the substituents are independently aryl, alkyl or
aralkyl, 2,2,2-trifluoroethyl, and the like. The preferred groups
for the protection of the hydroxyl side chain are adamantyl,
t-butyl, 4-methoxybenzyl, cyclopentyl and cyclohexyl.
When the side chain contains a phenol, such as in tyrosine, it may
be protected by such groups as methyl, methoxymethyl(MOM),
methoxyethoxymethyl(MEM), .beta.-trimethylsilylethyl,
methylthiomethyl, tetrahydropyranyl, isopropyl, cyclohexyl,
cyclopentyl, t-butyl, adamantyl, 4-methoxyphenylsilyl,
o-nitrobenzyl, 2,4-dinitrophenyl, m-bromobenzyl,
2,6-dichlorobenzyl, trisubstituted-silyl wherein the substituents
are independently alkyl, aryl or aralkyl, ethoxycarbonyl, carbamoyl
and the like. The most preferred protecting groups are adamantyl,
4-methoxybenzyl, t-butyl, cyclopentyl, and cyclohexyl. An
especially preferred protecting group is t-butyl.
A carboxy side chain, such as that found in aspartic acid or
glutamic acid, can be protected by the following groups: 1-or
2-adamantyl, methoxymethyl, methythiomethyl, t-butyl, methyl,
ethyl, phenyl, tetrahydropyranyl, cyclopentyl, cyclohexyl,
cycloheptyl, 4-picolyl, trisubstituted-silyl wherein the
substituents are independently alkyl, aryl or aralykl,
N-piperidinyl, N-succinimidoyl, .beta.-trimethylsilylethyl,
4-methoxybenzyl, benzyl, p-bromobenzyl, p-chlorobenzyl,
p-nitrobenzyl, phenacyl, N-phthalimidoyl,
4-alkyl-5-oxo-1,3-oxazolidinyl, trisubstituted-stannyl wherein the
substituents are independently alkyl, aryl or aralkyl, and the
like. The most preferred protecting group is t-butyl.
If the functional group on the side chain is mercapto, e.g.,
cysteine, such groups as tri-phenylmethyl, benzyl, 4-methylbenzyl,
3,4-dimethylbenzyl, 4-methoxybenzyl, .beta.-trimethylsilylethyl,
p-nitrobenzyl, 4-picolyl, diphenylmethyl, triphenylmethyl,
bis(4-methoxyphenyl)methyl, diphenyl-4-pyridylmethyl,
2,4-dinitrophenyl, t-butyl, t-butylthio, adamantyl,
isobutoxymethyl, benzylthiomethyl, thiazolidinyl, acetamidomethyl,
benzamidomethyl, 2-nitro-1-phenylethyl, 2,2-bis(carboethoxy)ethyl,
9-fluorenemethyl, acetyl, benzoyl, and the like can be used to
protect said group. In this case, it is preferred that the
protecting groups are t-butyl, t-butylthio, 4- methoxybenzyl, and
triphenylmethyl.
If the side chain contains an amino group, such as the
.epsilon.-amino group of lysine and ornithine, the following groups
may be used: 9-fluorenylmethyloxycarbonyl,
9-(2-sulfo)fluorenylmethyloxycarbonyl, .beta.-trimethylsilyl
ethyloxycarbonyl, 2-furanylmethyloxycarbonyl, adamantyloxycarbonyl,
carbobenzoxy, t-butyloxycarbonyl, t-amyloxycarbonyl,
cyclobutyloxycarbonyl, 1-methylcyclobutyloxycarbonyl,
cyclopentyloxycarbonyl, cyclohexyloxycarbonyl,
1-methylcyclohexyloxycarbonyl, isobornyloxycarbonyl, benzyl,
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
chlorobenzyloxycarbonyl, isonicotinyloxycarbonyl,
p-toluenesulfonylamidocarbonyl, methylsulfonylethyloxycarbonyl,
.beta.,.beta.,.beta.-trichloroethyloxycarbonyl, dithiasuccinoyl,
phthaloyl, 4,5-diphenyl-4-oxazoline-2-one, piperidino oxycarbonyl
trifluoroacetyl, chloroacetyl, p-toluenesulfonyl and the like.
Preferred groups for the protection of this amino side chain are
carbobenzoxycarbonyl, t-butyloxycarbonyl, and
adamantyloxycarbonyl.
If the amino acid has an imidazole group, such as in histidine, the
following groups may be used to protect the side chain:
benzyloxymethyl, piperdinylcarbonyl, phenacyl, pivaloyloxymethyl,
1-(alkoxycarbonylamino)- 2,2,2-trifluoroethyl,
1-trifluoromethyl-1-(p-chlorophenoxy-methoxy)-2,2,2-trifluoroethyl,
2,4-dinitrophenyl, toluenesulfonyl, FMOC, triphenylmethyl,
t-butyloxycarbonyl, t-butyloxymethyl and the like. The preferred
groups are FMOC, t-butyloxycarbonyl, triphenylmethyl, and
t-butyloxymethyl.
When the amino acid has a guanidine side chain, such as in
arginine, the following protecting groups can be used to protect
the .omega.-nitrogen on the guanidine moiety:
methoxytrimethylbenzenesulfonyl, pentamethylchromane-sulfonyl,
mesitylenesulfonyl, tolunesulfonyl, 2,4,6-trimethylbenzenesulfonyl,
trimethoxybenzensulfonyl, bisadamantyloxylcarbonyl, nitro, tosyl,
and the like. The preferred protecting groups are
methoxytri-methylsulfonyl, pentaamethylchromanesulfonyl,
bisadamantyloxycarbonyl, and mesitylenesulfonyl.
For side chains containing an amide group such as in glutamine and
asparagine, the following groups can be used to protect the side
chain: dimethoxybenzyhydryl, 9-xanthenyl, 2,4,6-trimethoxybenzyl,
and the like.
As used herein in the instant specification, the term "alkyl", when
used alone or in combination with other groups refers to a carbon
chain containing from 1 to 6 carbon atoms. They may be straight
chains or branched and include such groups as methyl, ethyl,
propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl,
amyl, n-hexyl and the like. The preferred alkyl group contains from
1-3 carbon atoms. The term aryl as used herein refers to an
aromatic ring system containing from 6-10 ring carbon atoms and up
to a total of 15 carbon atoms. It includes such groups as phenyl,
.alpha.-naphthyl, .beta.-naphthyl, and the like. The preferred
group is phenyl.
Aralkyl groups are aryl groups attached to the main chain through
an alkylene bridge. Such groups include benzyl, phenethyl and the
like. The preferred aralkyl group is benzyl.
The aryl and aralkyl groups herein may be unsubstituted or may be
substituted with an electron donating group in situations wherein
the protecting group is cleaved by acid. An electron donating group
as defined herein shall be interpreted as a group that will release
or donate electrons more than hydrogen would if it occupied the
same position in the molecule. See, J. Marsh, Advance Organic
Chemistry, 2nd Ed., McGraw Hill, Inc. (1977). These types of groups
are well known in the art. Examples of electron donating groups
include alkyl, lower alkoxy, aralkoxy; and the like. These electron
donating groups, e.g., alkoxy, may be present on the aryl moiety of
the following groups: DMB, TMB, Mtr, Pmc, Bz, Trt, CBZ and the
like.
The protecting groups described hereinabove are well known to one
skilled in the art. They can be removed under very mild acidic or
basic conditions. The preferred protecting groups are those which
can be cleaved by acid or base under conditions which are milder
than those used to cleave the benzyl group. These include groups
which can be cleaved by trifluoroacetic acid at room temperature
within one to four hours. The especially preferred protecting
groups are groups which can be cleaved by trifluoroacetic acid at
room temperature within 1-2 hours.
These protecting groups include such groups as tetrahydropyranyl,
.beta.-trimethylsilylethyl, 1-ethoxyethyl, t-butyl,
p-methoxybenzyl, 1-adamantyl, diphenylmethyl, triphenylmethyl,
trialkylsilyl, (e.g. tri-methylsilyl, triethylsilyl, and the like)
trialkylstannyl, (e.g. trimethylstannyl, triethylstannyl, and the
like), bis (4-methoxyphenyl)methyl, 2-furanylmethyloxycarbonyl,
t-amyl-oxycarbonyl, 1-methylcyclohexyloxycarbonyl, isobornyloxy
carbonyl, methoxytrimethylbenzenesulfonyl,
pentamethylchromanesulfonyl, 2,4,6-trimethoxybenzyl, 9-xantheneyl
and the like.
However, not all of the amino acids have sidechain functional
groups. For example, many amino acids have hydrogen, alkyl or
aralkyl side chains. These include glycine, alanine, valine
leucine, norleucine, phenylalanine, isoleucine and the like.
Therefore, these amino acids do not require protecting groups
thereon.
To differentiate between those amino acids having protecting groups
and those not having protecting groups thereon, the term ##STR29##
is used. As used herein, if no protecting group is present on the
amino acid side chain, such as, e.g., in alanine, (which doesn't
have a functional group and therefore no blocking group is
required) "X" is -. Moreover, if the amino acid side chain has a
functional group, such as in tyrosine, but is unprotected, then
this also is indicated by X being defined as -. In other words, in
both instances, when X is -, the side group is unprotected.
Although the term functional group is understood by one skilled in
the art, it is defined as a group which could react with the
reactants used or products formed under peptide forming condition
if not protected by a blocking group. These functional groups
include amino, carboxy, hydroxy, guanidine, imidazole, amino and
the like.
On the other hand, if X is a blocking group, then this signifies
that the functional group on the side chain is protected. For
example, if AA is serine and X is t-butyl, then the residue
##STR30## These protecting groups include the protecting groups
described hereinabove.
Abbreviations have been used in the specification and claims with
respect to these blocking groups and are listed hereinbelow:
______________________________________ Protecting group
Abbreviation ______________________________________
dimethoxybenzhydryl DMB 2,4,6-trimethoxybenzyl TMB
2,3,6-trimethyl-4 methoxybenzenesulfonyl Mtr
9-fluorenylmethyloxycarbonyl FMOC t-butoxycarbonyl BOC
t-butoxymethyl Bom pentamethylchromanesulfonyl Pmc adamantyl ada
.beta.-trimethylsilylethyl TMSE
.beta.-trimethylsilylethyloxycarbonyl TEOC t-butyl t-bu benzyl Bz
cyclopentyl Cp cyclohexyl Ch triphenylmethyl Trt benzyloxycarbonyl
Cbz adamantyloxycarbonyl Adoc formyl CHO trifluoroacetyl TFA
______________________________________
The term amino acid protecting group, as used herein, refers to
blocking groups which are known in the art and which have been
utilized to block the amino (NH.sub.2) group of the amino acid.
Blocking groups such as 9-lower
alkyl-9-fluorenyloxycarbonyl,2-chloro-1-indanylmethoxycarbonyl
(CLIMOC) and benz [f] indene-3-methyloxycarbonyl (BIMOC) and
dbd-TMOC are discussed in U.S. Pat. Nos. 3,835,175, 4,508,657,
3,839,396, 4,581,167, 4,394,519, 4,460,501 and 4,108,846, and the
contents thereof are incorporated herein by reference as is fully
set forth herein. Moreover, other amino protecting groups such as
2-(t-butyl sulfonyl)-2-propenyloxycarbonyl (Bspoc) and
benzothiophene sulfone-2-methyloxycarbonyl (Bsmoc) are discussed in
U.S. Pat. No. 5,221,754 and the subject matter therein is
incorporated herein by reference. Other amino protecting groups are
described in an article entitled "Solid Phase Peptide Synthesis" by
G. Barany and R. B. Merrifield in Peptides, Vol. 2, edited by E.
Gross and J. Meienhoffer, Academic Press, New York, N.Y., pp.
100-118 (1980), the contents of which are incorporated herein by
reference. These N-amino protecting groups include such groups as
the FMOC, Bspoc, Bsmoc, t-butyloxycarbonyl (BOC), t-amyloxycarbonyl
(Aoc), .beta.-trimethylsilylethyloxycarbonyl (TEOC),
adamantyloxycarbonyl (Adoc), 1-methylcyclobutyloxycarbonyl (Mcb),
2-(p-biphenylyl)propyl-2-oxycarbonyl (Bpoc),
2-(p-phenylazophenyl)propyl-2-oxycarbonyl (Azoc),
2,2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl (Ddz),
2-phenylpropyl-2-oxycarbonyl (Poc), benzyloxycarbonyl (Cbz),
p-toluenesulfonyl aminocarbonyl (Tac) o-nitrophenylsulfenyl (Nps),
dithiasuccinoyl (Dts), phthaloyl, piperidino- oxycarbonyl, formyl,
trifluoroacetyl and the like.
These protecting groups can be placed into four categories:
1) a base labile N.alpha.-amino acid protecting group such as FMOC,
and the like.
2) protecting groups removed by acid, such as Boc, TEOC, Aoc, Adoc,
Mcb, Bpoc, Azoc, Ddz, Poc, Cbz, 2-furanmethyloxycarbonyl (Foc),
p-methoxybenzyloxycarbonyl (Moz), Nps, and the like.
3) protecting groups removed by hydrogenation such as Dts, Cbz.
4) protecting groups removed by nucleophiles, such as Bspoc, Bsmoc
and Nps and the like.
5) protecting groups derived from carboxylic acids, such as formyl,
acetyl, trifluoroacetyl and the like, which are removed by acid,
base or nucleophiles.
As defined herein, a nucleophile is an electron-rich atom, i.e., an
atom which can donate an electron pair, which tends to attack a
carbon nucleus but does not act as a Bronsted Lowry base. For
example, a nucleophile, as defined herein, includes those molecules
which are used for nucleophilic addition across a double bond and
behaves in a manner similar to that described in the schemes herein
below.
The general mechanism for cleavage of Bspoc and Bsmoc groups are
similar in that the nucleophile is believed to react through a
Michael-type addition across a double bond. Although the following
schemes are shown for Bspoc, it is also illustrative of Bsmoc:
##STR31## The nucleophile is believed to attack at the terminal
carbon atoms of the propenyl group (Michael acceptor) forming a
zwitterion which can eliminate the OCOAA(X)OH anion and H+ to form
an alkene-amine and the carbamic acid (8) after protonation. Loss
of CO.sub.2 will furnish the free amino acid.
The nucleophiles which will function in concert with this invention
must have an active hydrogen atom, i.e., a hydrogen atom attached
to the nucleophilic atom.
It is preferred that the nucleophile is a simple amine. It is
especially preferred that the simple amine is a primary or
secondary amine of the formula HNR.sub.19 R.sub.20 wherein R.sub.19
and R.sub.20 are independently hydrogen, lower alkyl or substituted
lower alkyl, the lower alkyl being substituted with OH, CH.sub.3,
or CH.sub.2 CH.sub.3 or R.sub.19 and R.sub.20 taken together form a
mono or bicyclic ring containing from 4 to 10 ring carbon atoms and
1 or 2 heteroatoms selected from the group consisting of nitrogen,
sulfur or oxygen.
Typical examples of useful amines include ethanolamine, morpholine,
piperidine, diethylamine, 2,6-dimethylpiperidine, piperazine,
diethylamine, ethylamine and the like.
An organo mercaptan can also be used as a nucleophile, e.g., alkyl
mercaptans, cycloalkyl mercaptans, aryl mercaptan or aralkyl
mercaptans. The most preferred mercaptan is benzyl mercaptan.
However, when an organomercaptan is used as the nucleophile, the
deblocking reaction additionally requires a base catalyst, such as,
for example, triethylamine and the like.
The nucleophile can be added as a free compound or as an insoluble
reagent attached to a solid support i.e., polystyrene or silica
dioxide. These are represented by the formula:
wherein p is an organic polymer as defined hereinabove or a silica
gel polymer; alk is a chemical bond, alkyl or aroyl chain having
from about one to about ten carbon atoms and Nu-H is a nucleophile
as defined hereinabove.
A preferred insoluble reagent is the silica based piperazine
reagent 9: ##STR32##
Another useful nucleophile is benzylmercaptan as shown in the
following scheme. ##STR33## In this scheme the thio-group reacts in
a Michael fashion to remove the Bspoc protecting group.
The amino acid fluorides of the present invention can be prepared
by art recognized techniques. More specifically, they can be
prepared by reacting an N-protected amino acid with the reagent
cyanuric fluoride according to the following equation: ##STR34##
wherein BLK is an amino protecting group as defined herein and X is
defined herein. It is preferred that BLK is the FMOC CLIMOC, BIMOC,
DBD-TMOC, Bspoc, Bsmoc, or related base sensitive group. This
reaction can be run at temperatures as low as 0.degree. and up to
the refluxing temperature of the solvent, but it is preferred that
reaction is run at room temperature. It also can be run in an inert
solvent such as pyridine/CH.sub.2 Cl.sub.2 and the like.
The cyanuric fluoride can be prepared from the corresponding
chloride in the presence of potassium fluoride at elevated
temperatures ranging from 150.degree. to 250.degree. C., according
to the following equation: ##STR35##
Other fluorinating agents well known in the art, such as thionyl
fluoride, 2,4,6-trinitrofluorobenzene, N-methyl-2-fluoropyridinium
salts, and the like may be used in place of KF to effect the
formation of cyanuric fluoride.
Besides the methods described hereinabove, a new method has been
developed to synthesize the protected amino acid fluorides of the
instant specification. The new fluorinating agent is a
fluoro-formamidinium salt and has the formula: ##STR36## wherein
R.sub.15, R.sub.16, R.sub.17, and R.sub.18 are independently lower
alkyl or aryl, aryl lower alkyl, cycloalkyl, cylcoalkyl lower alkyl
or R.sub.15 and R.sub.16 taken together with the nitrogen atom to
which they are attached form a 5- or 6-membered ring
or R.sub.15, R.sub.16, R.sub.17 and .sub.18 taken together with the
nitrogen atom to which they are attached form a 5- or 6-membered
ring or
R.sub.15, R.sub.16, R.sub.17 and R.sub.18 taken together with the
nitrogen atom to which they are attached form a 5- or 6-membered
ring and
A is a counterion.
The aryl, arylalkyl and alkyl groups are as defined
hereinabove.
The term "cycloalkyl" refers to a single ring or a fused ring
system containing 3-10 ring carbon atoms and up to a total of 12
carbon atoms. The only ring atom in cycloalkyl are carbon atoms,
i.e., there are no hetero ring atoms. The cycloalkyl group may be
completely saturated or partially saturated. It may contain one
ring or it can contain two, three or more rings. It is preferred
that the cycloalkyl group be bicyclic and especially monocyclic. In
addition, it is preferred that the ring contain 5-10 ring carbon
atoms, especially, 5, 6 or 10 ring carbon atoms. Examples include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, adamantyl, decalinyl, indanyl, and the like. The
preferred cycloalkyl groups are cyclopentyl and cyclohexyl.
Cycloloweralkyl is an alkylene group, as defined above, bridging
the main chain with a cycloalkyl group, as defined herein. Examples
include cyclopentylmethyl, cyclohexylmethyl, and the like.
A counterion, as used herein, is an anion used to neutralize the
cationic portion of the molecule. Examples include
hexafluorophosphate, halide, sulfate, BF.sub.4.sup.-, sulfite,
nitrate, nitrite, acetate, phosphate, oleate, sulide, carboxylate,
bisulfate, and the like.
Preferred values of R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are
lower alkyl and aryl. It is preferred that R.sup.15, R.sub.16,
R.sub.17 and R.sub.18 are alkyl containing 1-3 carbon atoms,
especially methyl or phenyl. It is preferred that at least two and
more preferably, three of R.sub.15, R.sub.16 and R.sub.17 are
loweralkyl. Examples of the fluoroformamidinium salt of the present
invention include tetramethyl fluoroformamidinium
hexafluorophosphate (TFFH), trimethylphenylfluoroformamidinium
hexafluorophosphate (TPFFH), and the like.
It is also preferred that R.sub.17 and R.sub.18 and/or R.sub.15 and
R.sub.16 taken together with the nitrogen atom to which they are
attached form a 5- or 6-membered ring, e.g., piperidine,
pyrrolidine, and the like. It is even more preferred that both
R.sub.17 and R.sub.18 taken together with the nitrogen atom to
which they are attached and R.sub.15 and R.sub.16 taken together
with the nitrogen atom to which they are attached form a 5- or
6-membered ring. In both instances, the rings may be unsubstituted
or substituted with lower alkyl. It is even more preferred that the
ring that is formed between R.sub.15, R.sub.16 and the nitrogen
atom to which they are attached is the same and the ring formed
between R.sub.16 and R.sub.17 and the nitrogen atom to which they
are attached are the same. Examples thereof include
bis(tetramethylene) fluoroformamidinium, hexafluorophosphate
(BTFFH) and the like.
Another preferred embodiment of the present invention is when
R.sub.15, R.sub.16, R.sub.17 and R.sub.18 taken together form a 5-
or 6-membered ring, such as, for example, imidazolidine,
hexahydropyrimidine, and the like, which may be unsubstituted or
substituted with lower alkyl. An example thereof is 1,
3-dimethyl-2-fluoroimidazolium hexafluorophosphate (DFIH), and the
like.
The fluoroformamidinium salts of the present invention are prepared
in accordance with art recognized techniques. For example, the
fluoroformamidinium salts can be prepared from reacting the
corresponding chloride salt with a fluorinating agent such as a
fluoride, (e.g., alkali fluoride, such as KF, NaF, and the like) or
PF.sub.6.sup.-, and the like in a dry inert polar solvent, such as
acetonitrile, and the like. The reaction can be effected at
temperatures as low as 0.degree. and up to the refluxing
temperature of the solvent, but it is preferred that the reaction
is run at room temperature.
The chloro derivatives can be prepared in accordance with art
recognized techniques, such as the methodology described by
Dourtaglou and Gross in Synthesis 572 (1984), the contents of which
are incorporated by reference. For example, a urea derivative such
as ##STR37## is reacted with a chlorinating agent phosgene or
oxalyl chloride, and the like, which after CO.sub.2 evolution, and
addition of the counterion, affords the chloro formadinium salt,
wherein R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are as defined
herein.
The fluoroformamidinium salt of the present invention, as indicated
hereinabove, can also be used to synthesize the protected amino
acid fluorides of the present invention. More specifically, the
protected amino acid fluorides of the present invention can be
prepared by reacting an N-protected amino acid with the
fluoroformamidinium salt. This reaction is preferably run in an
inert solvent, such as chloroform or methylene chloride. This
reaction can be run at temperatures as low as 0.degree. C. and up
to the boiling point of the solvent, but it is preferred that the
reaction is run at room temperature. Additionally, it is preferred
that the reaction is run in the presence of a base, such as
pyridine, triethylamine, and the like.
The amino acid fluorides of the present invention are useful in
peptide bond formation. The scope is quite broad, as the amino acid
fluorides of the present invention can be coupled with an amino
acid, a dipeptide, tripeptide, or higher peptide, having a free
terminal amino group. As used herein, the term first amino acid is
meant to include amino acids as well as dipeptides, and the higher
peptides.
The synthesis of peptides according to the present invention
requires the following steps:
1) protection of the carboxyl group on a first amino acid.
2) formation of the amino acid fluorides of the present invention
in accordance with the procedure herein.
3) formation of the peptide bond by coupling the amino acid
fluoride with the first amino acid.
4) removal of the protecting groups.
A variety of carboxy protecting groups known in the art may be
employed. Examples of many of these possible groups may be found in
"Protective Groups in Organic Synthesis", by T. W. Green, John
Wiley & Sons, 1981, the contents of which is incorporated
herein by reference.
The following sequence is illustrative of the coupling of an amino
acid fluoride of the present invention with an amino acid having a
free amino group: ##STR38##
In the above scheme, BLK is as defined hereinabove, X.sub.1,
X.sub.2 and X.sub.3 are independently defined as X hereinabove, and
P is a carboxy protecting group, e.g., methyl ester, t-butylester,
.beta.-trimethylsilylethyl ester, benzyl ester and the like.
As shown by the above scheme, the N.alpha.-amino protected amino
acid fluoride is reacted with a second amino acid in which the
carboxy group is protected. A peptide is formed between the first
amino acid and the second amino acid. The peptide chain can be
increased by removing the alpha amino protecting group by
techniques known to one skilled in the art, and then reacting the
corresponding di-peptide with another N.alpha.-amino protected
amino acid fluoride to form the corresponding tri-peptide. The
N.alpha.-amino protecting group of the tri-peptide is removed and
the above cycle is repeated until the desired peptide has been
obtained.
The coupling of the N-.alpha. protected amino acid fluoride with
the carboxy protected amino acid by the normal two phase technique
takes place without racemization.
The present invention can readily be utilized in solid phase
peptide synthesis. Solid phase peptide synthesis is based on the
stepwise assembly of a peptide chain while it is attached at one
end to a solid support or solid phase peptide resin. Two methods
are generally well known in the art.
One, the Merrifield method, employs a solid support for attachment
of the amino acid or peptide residues. This method employs
N-protected amino acids as building blocks which are added to an
amino acid or peptide residue attached to the solid support at the
acyl (acid) end of the molecule. After the peptide bond has been
formed, the protecting group is removed and the cycle repeated.
When a peptide having the desired sequence has been synthesized, it
is then removed from the support.
The second method, the inverse Merrifield method, employs reagents
attached to solid supports in a series of columns. The amino acid
or peptide residue is passed through these columns in a series to
form the desired amino acid sequence.
These methods are well known in the art as discussed in U.S. Pat.
Nos. 4,108,846, 3,839,396, 3,835,175, 4,508,657, 4,623,484,
4,575,541, 4,581,167, 4,394,519 as well as in Advances in
Enzymology, 32, 221 (1969) and in PEPTIDES, VOL, 2, edited by
Erhard Gross and Johannes Meienhoffer, Academic Press, New York,
N.Y. York pp. 3-255 (1980) and are incorporated herein by reference
and is fully set forth herein.
During peptide synthesis, it may not be necessary to actually
isolate the amino acid fluorides. The protected amino acid fluoride
may be prepared in situ and then used in the coupling reaction with
the carboxy protected amino acid or peptide. The
fluoroformamidinium salts of the present invention permits the
skilled artisan to accomplish these goals.
More specifically, in addition, to its use as a source of protected
acid fluoride, the fluroformamidinium salts of the present
invention can be used as coupling agents for peptide synthesis in
which in situ formation of the intermediate acid fluoride precedes
the coupling. This approach avoids the need to isolate, purify, and
store the acid fluoride, yet allows one to take advantage of the
great reactivity of this class of coupling compounds. Furthermore,
under these circumstances, there is little, if any, racemization.
For example, the coupling reaction of Z-Phe-Val-OH with H-Ala-OMe
carried out with the use of tetramethylfluoroformamidinium
hexafluorophosphate, in the presence of proton sponge at
-30.degree. C. gives the expected tripeptide, with only about 1%
racemization.
In addition to its use in the synthesis of protected amino acid
fluorides and as a simple in situ coupling reagent, the
fluoroformamidinium salts of the present invention, such as TFFH,
can be used as a coupling agent for assembly of peptides by both
solution and solid phase techniques. An example is the synthesis of
leucine enkephalin H-Tyr-Gly-Gly-Phe-Leu-OH in 53% yield (88.5%
purity) on an automated peptide synthesizer (Millipore 9050). Two
other examples of solid phase syntheses involved synthesis of the
nonamer prothrombin and the 20-mer alamethicin acid,
H-Ala-Asn-Lys-Gly-Phe-Leu-Gly-Glu-Val-NH.sub.2 and
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gin-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Glu-Gln
-Phe-OH, respectively. The latter is unique in that it consists of
many hindered amino acids, including .alpha.-aminoisobutyric acid
(Aib), and could not previously be made by solid phase techniques
except via FMOC amino acid fluorides.
The coupling reaction may also contain other additives normally
utilized in peptide synthesis such as those utilized to prevent
racemization. For example, besides the fluoroformamidinium salts,
the following compounds may additionally be added to the coupling
reaction: ##STR39## and N-oxides thereof and salts thereof wherein
R.sub.1 and R.sub.2 taken together with the carbon atoms to which
they are attached form a heteroaryl ring wherein said heteroaryl
ring is an oxygen, sulfur or nitrogen containing heteroaromatic
containing from 3 and up to a total of 13 ring carbon atoms, said
heteroaryl may be unsubstituted or substituted with lower alkyl or
an electron-donating group;
Y is O, NR.sub.4, CR.sub.4 R.sub.5 ;
R.sub.5 is independently hydrogen or lower alkyl;
X is CR.sub.6 R.sub.7 or NR.sub.6 ;
R.sub.6 or R.sub.7 are independently hydrogen or lower alkyl; or
R.sub.6 and R.sub.7 taken together form an oxo group or when n=O,
R.sub.4 and R.sub.6 taken together may form a bond between the
nitrogen or carbon atom of Y and the nitrogen or carbon atom of
X;
Q is (CR.sub.8 R.sub.9) or (NR.sub.8);
when n is 1, R.sub.4 and R.sub.8 taken together may form a bond
between the ring carbon or nitrogen atom of Q and the ring carbon
or nitrogen atom of R.sub.8 ;
n is 0, 1 or 2;
R.sub.3 is hydrogen;
R.sub.8 and R.sub.9 are independently hydrogen or lower alkyl or
R.sub.7 and R.sub.8 taken together with the carbon to 95 which they
are attached form an aryl ring.
Examples include 1-hydroxy-7-azabenzotriazole,
1-hydroxy-4-aza-benzotriazole, 1
hydroxy-4-methoxy-7-azabenzotriazole
4-N,N-dimethylamino-1-hydroxy-7-aza-benzotriazole,
1-hydroxy-6-azabenzotriazole, 1-hydroxy-5-azabenzotriazole,
1-hydroxy-7-aza-1H-indazole, 1-hydroxyl-7-azabenzo-1H-imidazole,
1-hydroxy-1H-pyrrolo [2,3-b]pyridine,
1-hydroxy-4-t-butyl-7-azabenzotriazole, and the like. In addition,
1-hydroxy benzotriazole could also be utilized as the additive.
These compounds as well as other compounds of Formula II are
described in copending application U.S. Ser. No. 127,675, the
contents of which are incorporated by reference.
The amino acid fluorides of the present invention can exist in
various stereoisomeric forms, viz., the D or L stereoisomers.
Moreover, the amino acid fluorides may be present in mixture of the
D and L forms such as in racemic mixtures. All of these forms are
contemplated to be within the scope of the present invention. It is
preferred that the stereoisomer of the amino acid fluorides of the
present invention exist in the L form.
EXAMPLES
The invention will now be illustrated by examples. The examples are
not intended to be limiting of the scope of the present invention.
In conjunction with the general and detailed descriptions above,
the examples provide further understanding of the present
invention.
I PREPARATIVE EXAMPLES: PREPARATION OF CYANURIC FLUORIDE
A 250 mL three necked round bottomed flask equipped with a
thermometer, stirring bar, distilling apparatus and powder dropping
funnel was charged with oven-dried sodium fluoride (53 g, 1.25
mole) and tetramethylene sulfone (sulfolane, TMS) (145 g, 115 mL,
1.20 mole, d=1.26). To the suspension was added cyanuric chloride
(62 g. 0.33 mole) in small portions by use of the powder dropping
funnel over a 10-min period. The reaction mixture was gradually
heated to 250.degree. C. by means of a heating mantle. The product
distilled from the flask as formed and was collected in glass traps
cooled in a Dry Ice-acetone bath. Collection of the distillate (29
mL) continued until distillation stopped (bp 72.degree.-78.degree.
C.). Redistillation gave 24 mL (86.19 g, 38.4% d=1.6 g/mL) of the
fluoride as a clear colorless liquid, bp 72.degree.-73.degree. C.,
lit bp 74.degree. C.
II GENERAL PROCEDURE FOR PREPARATION OF
N-(9-FLUORENYL-METHOXYCARBONYL)AMINO ACID FLUORIDES:
A solution (or suspension) of FMOC-amino acid (1 mmole) in dry
CH.sub.2 Cl.sub.2 (5 mL) was kept under nitrogen and treated with
cyanuric fluoride (1.08 g, 8 mmol, 700 uL, d=1.6) and pyridine (81
uL, 1 mmole). A clear solution was obtained which was refluxed (or
stirred at room temperature) for 45-120 min. Completion of reaction
was checked by TLC. During the reaction a white precipitate
separated. The mixture was extracted with ice-water (2.times.15 mL)
which caused the precipitate to dissolve. The organic layer was
dried over MgSO.sub.4. Filtration and solvent removal gave a
residue (solid or oil) which was crystallized from CH.sub.2
Cl.sub.2 /hexane or Et.sub.2 O/hexane to give the corresponding
FMOC-N-protected amino acid fluoride as a white crystalline solid.
The crude and recrystallized acid fluorides were analyzed by HPLC
following the same technique described for the corresponding
chlorides except that it was necessary to wait for 15-300 min
following addition of the fluoride to dry methanol in order to
allow time for complete conversion to the methyl ester. For
example, a mixture initially analyzing for 93.45% FMOC-Gly-F (as
methyl ester) came to complete conversion after 50 min with a
measured content of 98.89% FMOC-Gly-OMe and 1.00% FMOC-Gly-OH. In
case of FMOC-Val-F, initially analyzing for 82.70% FMOC-Val-F (as
methyl ester) complete conversion occurred after 5 hours with a
measured content of 98.19% FMOC-Val-OMe and 0.94% FMOC-Val-OH.
Fischer esterification of the free acid in the methanolic HF
solution did not occur. For example, a mixture initially containing
52.27% of FMOC-Gly-OH showed no significant change after 15 hours
(52.19% acid) and the same results were observed in case of
FMOC-Val-OH.
Example 1
N-(9-Fluorenylmethoxycarbonyl)glycine Fluoride
The reaction was used in accordance with the procedure described
above.
Reaction was completed after 2 hours of reflux, the fluoride being
obtained in 80.5% yield as pale yellow needles, mp 140.1.degree.
C., (98.9% pure according to HPLC analysis); IR (KBr) 3337 (NH),
1843 (COF), 1680 (OCON) cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3)
.delta.4.1-4.3 (m, 3, CHCH.sub.2), 4.5 (d, 2, NCH.sub.2 CO), 5.3
(bs, 1, NH), 7.15-7.8 (m, 8, aryl).
Anal. Calcd for C.sub.17 H.sub.14 FNO.sub.3 : C, 68.22; H, 4.71; N,
4.67. Found: C, 68.25, H, 4.63; N, 4.79.
Example 2
N-(9-Fluorenylmethoxycarbonyl)alanine Fluoride
The reaction was run in accordance with the procedure described
hereinabove.
Reaction was complete after 2 hours of reflux, the fluoride being
obtained in 75.4% yield as a white solid, mp 111.degree.-2.degree.
C., (98.66% pure according to HPLC analysis); [.alpha.]D.sup.23
+3.6.degree. (c 0.5, EtOAc); IR (KBr) 3326 (NH), 1845 (COF), 1690
cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3) .delta.1.6 (d, 3, CH.sub.3),
4.2 (t, 1, CHCH.sub.2), 4.5 (m, 3, CH.sub.2 O, NCHCO), 5.2 (d, 1,
NH), 7.2-7.8 (m, 8, aryl).
Anal. Calcd for C.sub.18 H.sub.16 FNO.sub.3 : C, 69.00; H, 5.14; N,
4.47. Found: C, 69.16; H, 5.30; N, 4.30.
Example 3
N-(9-Fluorenylmethoxycarbonyl)valine Fluoride
The reaction was run in accordance with the procedure described
hereinabove.
Reaction was complete after 2 hours of reflux, the fluoride being
obtained in a yield of 70.2% as a white solid, mp
113.degree.-4.degree. C., (98.62% pure according to HPLC analysis);
[.alpha.]D.sup.24 +10.7.degree. (c 1, CH.sub.2 Cl.sub.2); IR (KBr)
3312 (NH), 1843 (COF), 1688 (OCON) cm.sup.-1 ; .sup.1 H NMR
(CDCl.sub.3) .delta.1.0 (d, 6, CH.sub.3), 2.3 (m, 1, CHCH.sub.2),
4.2 (t, 1, CHCH.sub.3), 4.5 (m, 3, CH.sub.2 0, NCHCO), 5.15 (d, 1,
NH), 7.2-7.8 (m, 8, aryl).
Anal. Calcd for C.sub.20 H.sub.20 FNO.sub.3 C, 70.36; H, 5.90; N,
4.10. Found: C, 70.27; H, 5.92; N, 4.19.
Example 4
N-(9-Fluorenylmethoxycarbonyl)leucine Fluoride
The reaction was run in accordance with the procedure described
above.
Reaction was complete after 1 hour of reflux, the fluoride being
obtained in a yield of 75.2% as a white solid, mp
95.5.degree.-6.5.degree. C., (98.58% pure according to HPLC
analysis); [.alpha.]D.sup.23 -5.8.degree. (c 1, EtOAc); IR (KBr)
3336 (NH), 1938 (COF), 1699 (OCON) cm.sup.-1 ; .sup.1 H NMR
(CDCl.sub.3) .delta.1.00 (d, 6, CH.sub.3), 1.6-1.8 (m, 3, CH.sub.2
CH), 4.2 (t, 1, CHCH.sub.2 O), 4.5 (m, 3, CH.sub.2 O, NCHCO), 5.1
(d, 1, NH), 7.2-7.8 (m, 8, aryl).
Anal. Calcd for C.sub.21 H.sub.22 FNO.sub.3 : C, 70.96; H, 6.23; H,
3.94. Found: C, 70.70; H, 6.48; N, 4.15.
Example 5
N-(9-Fluorenylmethoxycarbonyl)isoleucine Fluoride
The reaction was run in accordance with the procedure described
above.
Reaction was complete after 1.5 hours of reflux, the fluoride being
obtained in a yield of 73.3% as a white solid, mp
115.degree.-6.degree. C., (97.13%) pure according to HPLC
analysis);
[.alpha.]D.sup.23 +15.6.degree. (c 0.5, EtOAc); IR (KBr) 3304 (NH),
1840 (COF), 1996 (OCON) cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3)
.delta.1.00 (m, 6, CH(Me)CH.sub.2 Me), 1.1-1.6 (m, 2, CHCH.sub.2
CH.sub.3), 2.00 (m, 1, CH), 4.2 (t, 1, CHCH.sub.2 O), 4.5 (m, 3,
CH.sub.2 O, NCHCO), 5.2 (d, 1, NH), 7.2-7.8 (m, 8, aryl).
Analy. Calcd for C.sub.21 H.sub.22 FNO.sub.3 : C, 70.96; H, 6.23;
N, 3.94. Found: C, 68.30; H, 6.06; N, 3.87.
Example 6
N-(9-Fluorenylmethoxycarbonyl)proline Fluoride
The reaction was run in accordance with the procedure described
above.
Reaction was complete after 12 hours of stirring at room
temperature, the fluoride being obtained in a yield of 78.2% as a
white solid mp, 88.degree.-9.degree. C.; [.alpha.]D.sup.30
-28.6.degree. (c 0.5, EtOAc).
Anal. Calcd for C.sub.20 H.sub.18 FNO.sub.3 : C, 70.78; H, 5.34; N,
4.12. Found: C, 70.86; H, 5.43, N, 4.21.
Example 7
N-(9-Fluorenylmethoxycarbonyl)phenylalanine Fluoride
The reaction was run in accordance with the procedure described
above.
Reaction was complete after 1 hour of reflux, the fluoride being
obtained in a yield of 63.9% as white crystals, mp
118.degree.-20.degree. C., (99.3% pure according to HPLC analysis);
[.alpha.]D.sup.24 +35.5.degree. (c 1, CH.sub.2 Cl.sub.2); IR (KBr)
3318 (NH), 1843 (COF), 1700 (OCON) cm.sup.-1 ; .sup.1 H NMR
(CDCl.sub.3) .delta.3.2 (d, 2, CH.sub.2 C.sub.4 H.sub.5), 4.2 (t,
1, CHCH.sub.2 O), 4.45 (m, 2, CH.sub.2 O), 4.85 (m, 1, NCHCO), 5.1
(d, 1, NH), 7.1-7.8 (m, 13 aryl).
Anal. Calcd for C.sub.24 H.sub.20 FNO.sub.3 : C, 74.03; H, 5.14; N,
3.59; F, 4.88. Found: C, 74.03; H, 5.13; N, 3.69; F, 4.68.
Example 8
N-(9-Fluorenylmethoxycarbonyl)tryptophan Fluoride
The reaction was run in accordance with the procedure described
above.
Reaction was complete after 1 hour of stirring at room temperature,
the fluoride being obtained in a yield of 70.7% as a white solid,
mp 125.degree.-8.degree. C. (dec.) (98.2% pure according to HPLC
analysis); [.alpha.]D.sup.24 -5.2.degree. (c 1, EtOAc); IR (KRr)
3390 and 3360 (NH), 1845 (COF), 1697 (OCON) cm.sup.-1, .sup.1 H NMR
(CDCl.sub.3) .delta.3.4 (d, 2, CH.sub.2 CHCO), 4.2 (t, 1,
CHCH.sub.2 O), 4.4 (d, 2, CHCH.sub.2 O), 4.9 (m, 1, NCHCO), 5.3 (d,
1, NH), 7.0-8.2 (m, 14, NH+aryl).
Anal. Calcd for C.sub.33 H.sub.31 FNO.sub.3 : C, 72.88; H, 4.94; N,
6.53. Found: C, 72.83, H, 5.01; N, 6.43.
Example 9
N-(9-Fluorenylmethoxycarbonyl)-O-(t-Butyl)serine Fluoride
The reaction was run in accordance with the procedure described
above.
Reaction was completed after 1 hour of stirring at room
temperature, the fluoride being obtained in a yield of 72.7% as
white crystals, mp 89.degree.-91.degree. C., (98.26% pure according
to HPLC analysis); [.alpha.]D.sup.26 +28.8.degree. (, 5, EtOAc); IR
(KBr) 3444 (NH), 1868 (COF), 1733 (OCON) cm.sup.-1 ; .sup.1 H NMR
(CDCl.sub.3) .delta.1.2 (s, 9, CMe.sub.3), 3.6 (q, 1, CHHOCMe), 3.9
(q, 1, CHHOCMe.sub.3), 4.22 (t, 1, CHCH.sub.2 OCO), 4.45 (m, 2,
CH.sub.2 OCO), 4.7 (m, 1, NCHCO), 5.65 (d, 1, NH), 7.25-7.8 (m, 8,
aryl).
Anal. Calcd for C.sub.22 H.sub.24 FNO.sub.4 : C, 68.55; H, 6.27; N,
3.63. Found: C, 68.49; N, 6.32; N, 3.67.
Example 10
N-(9-Fluorenylmethoxycarbonyl)-O-(t-Butyl)threonine Fluoride
The reaction was run in accordance with the procedure described
hereinabove:
Reaction was complete after 1 1/2 hours of stirring at room
temperature, the fluoride being obtained in a yield of 72.6% as
white crystals, mp 53.5.degree. C., (98.03% pure according to HPLC
analysis); [.alpha.]D.sup.27 +12.3.degree. (c 0.4 EtOAc); IR (KBr)
3320 (NH), 1857 (COF), 1726 (OCON) cm.sup.-1 ; .sup.1 H NMR
(CDCl.sub.3) .delta.1.15 (s, 9, CMe.sub.3), 1.3 (d, 3, CH.sub.3),
4.3-4.5 (m, 5, CHCH.sub.2 OCO, NCH(CHO--)CO), 5.6 (d, 1, NH),
7.2-7.8 (m, 8, aryl).
Anal. Calcd for C.sub.23 H.sub.22 FNO.sub.4 : C, 69.15; H, 6.56; N,
3.50. Found: C, 69.11; H, 6.83; H, 4.00.
Example 11
N -(9-Fluorenylmethoxycarbonyl)-N.epsilon.-(t-Butyloxy
carbonyl)-lysine Fluoride
The reaction was run in accordance with the procedure described
above.
Reaction was completed after 1 hour of stirring at room
temperature, the fluoride being obtained in a yield of 65.9% as
white crystals, mp.sub.23 128.degree.-30.degree. C. (99.5% pure
according to HPLC analysis); [.alpha.]D.sup.23 -2.2.degree. (c 0.5,
CH.sub.2 Cl.sub.2); IR (KBr) 2254 (NH), 1854 and 1836 (COF), 1693
(OCON) cm.sup.-1 ; .sup.1 H (CDCl.sub.3) 1.4 (s, 9, CMe.sub.3),
1.5-2 (m, 6, CH.sub.2 --CH.sub.2 --CH.sub.2), 3.15 (m, 1, CH.sub.2
NH), 4.2 (t, 1, CHCH.sub.2 O), 4.4-4.6 (m, 4, CH.sub.2 O, NCHCO,
CH.sub.2 NH), 5.7 (d, 1, NH), 7.2-7.8 (m, 8, aryl).
Anal. Calcd For C.sub.28 H.sub.31 FN.sub.2 O.sub.5 : C, 66.36; H,
6.64; H, 5.95. Found: C, 66.16; H, 6.50; N, 5.92.
Example 12
N-(9-Fluorenylmethoxycarbonyl)aspartic Acid
Fluoride-.beta.-(t-Butyl) Ester
The reaction was run in accordance with the procedure described
above.
Reaction was completed after 30 minutes of stirring at room
temperature, the fluoride being obtained in a yield of 67.8% as
white crystals, mp 74.degree.-5.degree. C., (97.97% pure according
to HPLC analysis); [.alpha.]D.sup.23 +4.00.degree. (c 0.5, EtOAc);
IR (KBr) 3320 (NH), 1856 (COF), 1725 (COO), 1695 (OCON) cm.sup.-1,
.sup.1 H NMR (CDCl.sub.3) .delta.1.45 (s, 9, CMe.sub.3), 2.9 (dq,
2, CH.sub.2 COO), 4.3 (t, 1, CHCH.sub.2 OCO), 4.5 (m, 2, CHCH2O),
4.85 (m, 1, HCHCO), 5.85 (d, 1, NH), 7.2-7.8 (m, 8, aryl).
Anal. Calcd for C.sub.23 H.sub.24 FNO.sub.5 : C, 66.81; H, 5.85; N,
3.38. Found: C, 67.03; H, 5.95; N, 3.70.
The results of the above synthesis are summarized in the Table
hereinbelow:
TABLE 1 ______________________________________ Synthesis of
FMOC-Amino Acid Fluorides Compound Yield % mp(.degree.C.)
[.alpha.].sub.D.sup.t.degree.C.
______________________________________ FMOC--Gly--F 80.5 140-1
FMOC--Ala--F 75.4 111-2 +3.6.degree. (c 0.5, EtOAc, 23)
FMOC--Val--F 70.2 113-4 +10.7.degree. (c 1, CH.sub.2 Cl.sub.2, 24)
FMOC--Leu--F 75.2 95-6 -5.8.degree. (c 1, EtOAc, 23) FMOC--Ile--F
73.3 115-6 +15.6.degree. (c 0.5, EtOAc, 23) FMOC--Phe--F 63.9
118-20 +35.5.degree. (c 1, CH.sub.2 Cl.sub.2, 24) FMOC--Trp--F 70.7
125-8 -5.2.degree. (c 1, EtOAc, 24) FMOC--Ser(tBu)--F 72.7 89-91
+28.8.degree. (c 0.5, EtOAc, 26) FMOC--Thr(tBu)--F 72.6 53-5
+12.3.degree. (c 0.4, EtOAc, 27) FMOC--Lys(BOC)--F 65.9 128-30
-2.2.degree. (c .5, CH.sub.2 Cl.sub.2, 24) FMOC--Asp(OtBu)--F 67.8
74-5 +4.0.degree. (c 0.5, EtOAc, 23)
______________________________________
These results indicate that cyanuric fluoride is suitable not only
for the preparation of simple FMOC-amino acid fluorides but also
for those containing t-BOC, t-Bu or CBZ-groups on the side chain.
The amino acid fluorides described hereinabove were obtained in
crystalline form.
The use of amino acid fluorides of the present invention being used
as peptide coupling agents is illustrated hereinbelow.
In the following example, the FMOC amino acids were utilized:
Example 13
USE OF FMOC-AA-F IN PEPTIDE SYNTHESIS
I General Method for Executing Rapid FMOC/4-AMP or FMOC/TAEA
Peptide Synthesis. Five millimeters of a 0.1M solution of the
C-terminal starting amino acid ester, 5 mL of CHCl.sub.3 containing
0.75 mmol of the appropriate ##STR40## (wherein Y is halo, i.e.,
chloro or fluoro) and 5 mL of 5% Na.sub.2 CO.sub.3 solution were
combined and the two phase mixture stirred vigorously for 10
minutes. The organic phase was separated and treated with 5 mL of
4-AMP or TAEA. After 30 minutes 40 mL of CHCl.sub.3 was added and
the organic phase washed with five 25-mL portions of 10% phosphate
buffer of pH 5.5. Alternatively two 25-mL portions of saturated
NaCl solution was used to remove excess 4-AMP or TAEA prior to
extractions with the phosphate buffer. The ozganic phase was
concentrated in vacuo to a volume of 5 mL on a rotary evaporator
and the resulting CHCl.sub.3 solution used analogously for
treatment with the next FMOC amino acid halide.
II Preparation of
FMOC-Val-Asp(CMe.sub.3)-Val-Leu-Leu-Ser(CMe).sub.3
-Tyr(CMe.sub.3)-OCMe.sub.3. The general procedure described above
was followed beginning with 330 mg (1 mmol) of
H-Tyr-(CMe.sub.3)-OCMe.sub.3 HCl, 462 mg (1.2 mmol) of
FMOC-Ser-(CMe.sub.3)-F, 10 mL of methylene dichloride and 10 mL of
5% sodium carbonate solution. The mixture was stirred for 15
minutes, the organic layer separated and treated with 7.5 mL of
TAEA for 30 minutes. The solution was washed with three 10-mL
portions of said sodium chloride solution and three 15-mL portions
of phosphate buffer (pH 5.5). The organic layer was then treated in
the same way in sequence with the remaining FMOC amino acid halides
(FMOC-Leu-Cl twice, FMOC-Val-Cl, FMOC-Asp(CMe .sub.3)-F and
FMOC-Val-Cl a second time). Evaporation of the organic layer was
followed by column chromatography to give the protected
heptapeptide, mp 249.degree.-251.degree. C., in 50-60% yield MSFAB:
1255 (M+1); calculated 1253.8 (M).
Example 14
Coupling of FMOC-Phe-F with H-Leu-OMe. Five millimeters of a 0.1M
solution of H-Leu-OMe in CHCl.sub.3 was treated with 5 mL of
CHCl.sub.3 containing 0.6 mmol of FMOC-Phe-F (mp
118.degree.-120.degree. ) and 5 mL of 5% Na.sub.2 CO.sub.3
solution. The two-phase mixture was stirred vigorously for 10
minutes, the layers separated and the organic layer dried over
MgSO.sub.4 and evaporated with a rotary evaporator. Without
purification the crude residue (90%) was examined by HPLC analysis
which showed no evidence for the DL-diastereomer (0.1%). The two
diasteromers are readily separated with a mobile phase consisting
of 1% isopropyl alcohol in hexane (retention times: LL, 13.26
minutes; DL-17.13 minutes at flow rate 1.5 mL/minutes) using a
Waters Radial Pak normal silica gel column 10 u, Z-module
fitting.
Example 15
CBZ-alanine Fluoride
CBZ-Ala (1 mmole) in dry CH.sub.2 Cl.sub.2 is kept under nitrogen
and treated with cyanuric fluoride (8 mmol) and pyridine (1 mmole)
to form the corresponding CBZ-Ala-F.
Example 16
N-formyl-O-(t-butyl) serine Fluoride
N-formyl serine (1 mmole) in dry CH.sub.2 Cl.sub.2 under nitrogen
is treated with cyanuric fluoride (8 mmol) and pyridine (1 mmol) to
form the above-identified product.
Example 17
2-(t-butylsulfonyl)-2-propenyl-oxycarbonyl-1-phenylalanine
Fluoride
A. t-Butyl Allyl Sulfide
To a solution of 350 mL of anhydrous ethanol maintained under
nitrogen was slowly added 22.99 g (1 mol) of sodium spheres. The
sodium dissolved within 90 minutes, and to the resulting sodium
ethoxide solution was added 90.19 g (1 mol) of t-butyl mercaptan
with mechanical stirring. Allyl bromide (120.98 g; 1 mol) was then
added dropwise to the mechanically stirred sodium t-butyl thiolate
solution. After the addition was complete, the mixture was refluxed
for 10 minutes, the solution allowed to cool, the precipitated
sodium bromide filtered, and the ethanol removed by distillation at
atmospheric pressure. The residue was diluted with 200 mL of water,
and the layers separated. The aqueous layer was extracted with five
40-mL portions of ether. The combined organic layers were extracted
with 150 mL of water, the organic layer dried over MgSO.sub.4,
filtered, and the solvent removed in vacuo from a water bath at
45.degree. C. to give a yellow liquid. Distillation through a
0.8.times.15-cm fractionating column gave 56.16 g (45%) of the
sulfide as a colorless liquid, bp 139.degree.-141.degree. C.
B. 1,3-Dibromo-2-(t-butylsulfonyl) Propane
To a stirred solution of 17.37 g (0.13 mol) of t-butyl allyl
sulfide in 133 mL of CCl.sub.4 at -24.degree. C. (CCl.sub.4 /dry
ice) was added dropwise a solution of 21.33 g (0.13 mol) of
Br.sub.2 in 67 mL of CCl.sub.4. A yellow solid precipitated during
the addition. The mixture was warmed to room temperature and
stirred for 10 minutes following complete solution of the yellow
solid. The resulting solution was poured into a mixture of 55.50 g
(0.27 mol) of 85% m-chloroperbenzoic acid in 490 mL of CH.sub.2
Cl.sub.2 kept at -24.degree. C., and the mixture stirred for 30
minutes at this temperature. The cooling bath was then removed and
the mixture stirred at room temperature overnight. The precipitated
m-chlorobenzoic acid was filtered and the filtrate washed with
three 200-mL portions of saturated NaHCO.sub.3, followed by 200 mL
of water. The organic layer was dried over MgSO.sub.4, filtered,
and the solvent removed in vacuo from a water bath at 45.degree. C.
The crude product was recrystallized from 20% EtOAc/Skelly B to
give 32.02 g (75%) of the dibromide, mp 139.degree.-140.degree.
C.
C. 2-(t-Butylsulfonyl)-2-propenyl Bromide
A mixture of 16.78 g (0.052 mol) of 1,3-dibromo-2-(t-butylsulfonyl)
propane and 14 mL (0.12 mol) of 2,6-lutidine in 55 mL of CH.sub.2
Cl.sub.2 was refluxed for 75 minutes. The solution was allowed to
cool to room temperature and extracted with three 80-mL portions of
5% HCl followed by 80 mL of water. The organic layer was dried over
MgSO.sub.41 filtered, and the solvent removed in vacuo from a water
bath at 45.degree. C. to give 11.46 g (91%) of the allyl bromide as
a white solid, mp 40.5.degree.-42.0.degree. C., which was used
without further purification.
D. 2-(t-Butylsulfonyl-2-propenyl Alcohol
A mixture of 8.55 g (35.3 mmol) of 2-(t-butylsulfonyl)-2-propenyl
bromide and 5.31 g (78.1 mmol) of sodium formate in 150 ml of
methanol was refluxed overnight. The solution was allowed to cool
and concentrated to 50 mL with the aid of a water aspirator,
resulting in the precipitation of excess sodium formate. The
residue was diluted with 150 mL of water and extracted with five
50-mL portions of CH.sub.2 Cl.sub.2. The organic layer was dried
over MgSO.sub.4, filtered, and the solvent removed in vacuo from a
water bath at 45.degree. C. The crude product was recrystallized
from 15% EtOAc/Skelly F to give 4.30 g (68%) of the alcohol as a
colorless solid, mp 53.5.degree.-54.5.degree. C.
E. 2-(t-Butylsulfonyl)-2-propenyl Chloroformate
To a solution of 6.67 g (37.4 mmol) of
2-(t-butylsulfonyl)-2-propenyl alcohol in 27 mL of dry THF at
0.degree. C. was added in one portion 27 mL of phosgene. The
solution was stirred for 1 hour at 0.degree. C. and allowed to
stand at room temperature overnight. Excess phosgene and solvent
were removed under reduced pressure. The crude product was
recrystallized from 25% ether/Skelly B to give 8.23 g (91%) of the
chloroformate as a colorless solid, mp 56.5-57.7.
F. 2-(t-Butylsulfonyl)-2-propenyloxycarbonyl-L-phenyl-alanine
A solution of 4.57 g (19.0 mmol) of 2-
(t-butyl-sulfonyl)-2-propenyl chloroformate and 5.64 g (18.6 mmol)
of t-butyl L-phenylalaninate hydrophosphite in 90 mL of CH.sub.2
Cl.sub.2 was stirred in the presence of 165 mL of 5% NaHCO.sub.3 at
room temperature for 2 hours. The aqueous phase was separated, and
the organic phase washed with three 75-mL portions of 5% HCl. The
organic phase was dried over MgSO.sub.4, filtered, and the solvent
removed in vacuo from a water bath at 45.degree. C. The resulting
oil was dissolved in 36 mL of 50% CH.sub.2 Cl.sub.2
/trifluoro-acetic acid, and the solution stirred at room
temperature for two hours. Excess trifluoracetic acid and solvent
were removed in vacuo from a water bath at 150.degree. C. The
resulting oil was crystallized from ether/Skelly F to give
approximately 6.25g (91%) of the colorless acid, mp
88.0.degree.-89.5.degree. C.
G. 2-(t-butylsulfonyl)-2-propenyloxycarbonyl-L-phenyl-alanine
fluoride
The product formed hereinabove in Example 17F (1 mmol) is placed in
dry CH.sub.2 Cl.sub.2 under nitrogen and treated with cyanuric
fluoride (8 mmol) and pyridine (1 mmol) to form the
above-identified product.
Example 18
BOC-Phe-F
BOC-Phe (1 mmole) in dry CH.sub.2 Cl.sub.2 under nitrogen is
treated with cyanuric fluoride (8 mmol) and pyridine (1 mmole) to
form the above-identified product.
Example 19
Coupling of Bspoc-Phe-F with H-Leu-OMe
Five milliliters of 0.1M solution of H-Leu-OMe in CHCl.sub.3 was
treated with 5 ml of CHCl.sub.3 containing 0.6 mmol of Bspoc-phe-F
and 5 ml of 5% Na.sub.2 CO.sub.3 solution. The two phase mixture is
stirred vigorously for 10 minutes, the layers separated and the
organic layer dried over MgSO.sub.4 and evaporated to afford the
coupled dipeptide.
Example 20
BIMOC-Phe-F
A. 1-Bromo-2-bromomethylnaphthalene
To a solution of 103 g of 1-bromo-2-methylnaphthalene (bp
98.degree.-120.degree. /0.5 mm, prepared in 90% yield by the method
of Adams and Binder in JACS, 63, 2771(1941) and 82 g or
N-bromosuccinimide in 1030 mL of CCl.sub.4 was added 0.54 g of
dibenzoyl peroxide. The reaction mixture was refluxed with stirring
for 3 h. After another 0.54 g of dibenzoyl peroxide was added, the
mixture was refluxed for 3 hours. The solution was allowed to stand
at room temperature overnight, and the resulting suspension was
brought to the boiling point and filtered while hot. Concentration
of the filtrate gave 139 g (99.4%) of the bromide as a light yellow
solid, mp 104.degree.-107.degree. C., which was recrystallized from
CCl.sub.4 to give 128 g (91.6%) of pure
1-bromo-2-bromomethylnaphthalene as colorless crystals, mp
106.5.degree.-107.5.degree. C.
B. Diethyl 2-(1-Bromo-2-naphthylmethyl)malonate
To a solution of NaOEt prepared from 9.66 g of sodium and 210 mL of
dry EtOH was added 63.76 mL of diethyl malonate and the reaction
mixture refluxed for 2 h. To the resulting yellow solution was
added in small portions 126 g of 1-bromo-2-bromomethylnaphthalene
and the reaction mixture refluxed for 16 h. Distillation of ethanol
from an oil bath (100.degree.-110.degree. C.) through a simple
Claisen head gave a yellow suspension, to which was added 350 mL of
CH.sub.2 Cl.sub.2 and 350 mL of H.sub.2 O. The aqueous layer was
extracted twice with 120-mL portions of CH.sub.2 Cl.sub.2, and the
combined organic layer was washed three times with 100-mL portion
of H.sub.2 O, dried (MgSO.sub.4) and evaporated to give 152 g
(95.6%) of the ester as a yellow solid, mp 60.degree.-65.degree. C.
Recrystallization from acetic acid gave 137 g (86.2%) of the pure
diester as a colorless solid, mp 77.degree.-79.degree. C.
C. .beta.-(1-Bromo-2-naphthyl)propionic Acid
To a solution of 160 g of the product formed in B hereinabove in
239 mL of methanol was added 538 mL of 5N NaOH solution. The
mixture was refluxed for 135 min, and cooled by means of an ice
bath to 0.degree. C. To the reaction mixture was added 320 mL of
ice water, and the resulting precipitate was collected by
filtration and washed several times with small portions of ice
water. To the ice-cold, stirred filtrate was added dropwise 5N HCl
solution until the solution was weakly acidic. The precipitate was
filtered and washed several times with small portions of water.
Drying gave 86 g (63.1%) of crude diacid as a light yellow solid,
mp 154.degree.-157.degree. C. Recrystallization from water gave 80
g (58.7%) of pure diacid as colorless crystals, mp
157.degree.-159.degree. C. A suspension of 47.5 g of the crude
malonic acid in 968 mL of 6N HCl solution was refluxed for 16 h.
The mixture was cooled by means of an ice bath to 0.degree. C.,
treated with 650 mL of CH.sub.2 Cl.sub.2, and stirred for 15 min.
The aqueous layer was extracted twice with 100-mL portions of
water, dried (MgSO.sub.4), and evaporated to give 30.3 g (74%) of
yellow solid, mp 116.degree.-119.degree. C., which was
recrystallized from alcohol to give 28 g (68%) of the acid as
colorless crystals, mp 123.degree.-124.degree. C.
D. .beta.-(1-Bromo-2-naphthyl)propionyl chloride
To a solution of 40.16 g of the product formed in C hereinabove in
802 mL of CH.sub.2 Cl.sub.2 was added 19.3 mL of thionyl chloride.
The mixture was refluxed for 4 h, cooled to room temperature, and
the solvent evaporated from a water bath (40.degree.-50.degree. C.)
with a rotary evaporator (10 mm) to give a red-brown residue. In
order to remove traces of thionyl chloride, small portions of
CH.sub.2 Cl.sub.2 were added and the solution reevaporated three
times. Eventually the crude acid chloride was obtained as a
red-brown oil. The crude product was used immediately for the next
step without further purification.
E. 4-Bromobenz[f]indan-1-one
To an ice cold, stirred solution of the above crude
.beta.-(1-bromo-2-naphthyl)propionyl chloride in 802 mL of dry
CH.sub.2 Cl.sub.2 was added 26.11 g of anhydrous AlCl.sub.3
carefully. The reaction mixture was refluxed for 2 h, cooled to
room temperature and treated carefully while stirring with 900 mL
of ice-water followed by 75 mL of conc. HCl. The brown precipitate
was filtered and washed five times with small portions of CH.sub.2
Cl.sub.2. The aqueous layer was separated and extracted twice with
100-mL portions of CH.sub.2 Cl.sub.2. The combined CH.sub.2
Cl.sub.2 extracts were washed three times with small portions of
H.sub.2 O, dried (MgSO.sub.4) and evaporated from a water bath
(50.degree.-60.degree. C.) with a rotary evaporator (7 mm) to give
30.1 g (80.1%) of the crude ketone as a yellow solid, mp
146.degree.-149.degree. C., which could be recrystallized from
acetic acid to give 28.5 g (76%) of the pure ketone as colorless
crystals, mp 149.degree.-151.degree. C.
F. Benz[f]indan-1-ol
To an ice-cold solution of 22 g of the product formed in E
hereinabove in 150 mL of dry THF was added carefully in small
portions 16.1 g of LiAlH.sub.4. Subsequently, another 270 mL of dry
THF was added to the suspension and the mixture was refluxed for 8
days. The reaction mixture was cooled to 0.degree. C. by means of
an ice-bath and treated dropwise with 50 mL of ice-water followed
by 1080 mL of 10% H.sub.2 SO.sub.4 solution. The mixture was
extracted three times with 100-mL portions of ether and the
combined ether extracts washed three times with 150-mL portions of
H.sub.2 O, dried (MgSO.sub.4), and evaporated from a water bath
(60.degree.-70.degree. C.) with a rotary evaporator (10 mm) to give
9.2 g (59.3%) of the crude alcohol as a light yellow solid, mp
135.degree.-139.degree. C. Recrystallization from benzene (45 mL)
gave 8.6 g (55.4%) of pure alcohol as colorless crystals, mp
139.degree.-141.degree. C.
G. Benz[f]indene
A solution of 8.6 g of benz[f]indan-1-ol in 250 mL of 10% H.sub.2
SO.sub.4 was refluxed for 24 h. After cooling to room temperature
the reaction mixture was extracted with three 150-mL portions of a
mixture of benzene and hexane (1:2). The extracts were washed three
times with 100-mL portions of water, dried (MgSO.sub.4), and
evaporated to give 7.6 g (98%) of a colorless solid, mp
160.degree.-163.degree. C. Recrystallization from 340 mL of 95%
ethanol gave 6.6 g (85%) of the hydrocarbon as colorless crystals:
mp 163.degree.-164.degree. C.
H. Benz[f]indene-1-methanol
A 1.0M solution of n-butyllithium (44 mL, 44 mmoles) was added
dropwise under a nitrogen atmosphere to a stirred solution of 5 g
(30.12 mmoles) of benz[f]indene in 140 mL of anhydrous ether and 20
mL of anhydrous THF cooled by means of a Dry Ice-acetone bath to
-70.degree. C. The temperature of the reaction mixture was not
allowed to exceed -50.degree. C. Benzindenyl lithium soon started
to precipitate as small red crystals. After completion of the
addition (about 2 hours), the reaction mixture was stirred at
-70.degree. C. for another 45 minutes before introduction of
formaldehyde. Paraformaldehyde, 13.5 g. dried overnight in vacuum
over phosphorus pentoxide, was stirred and heated in a dry flask
placed in an oil bath at 175.degree.-195.degree. C. The
formaldehyde gas was led through a 7-mm glass tube into the
benz[f]indenyl lithium solution (held below -50.degree. C.) by a
stream of dry nitrogen. The temperature was not allowed to exceed
-50.degree. C. After completion of the addition, 280 mL of 10% HCl
solution was slowly poured into the stirred reaction mixture. The
mixture was stirred for 15 minutes at room temperature. After the
ether layer was separated, the aqueous solution was extracted twice
with 50-mL portions of ether and the combined ether solution was
washed with small portions of water until neutral. The ether
solution was dried (MgSO.sub.4) and evaporated to give a light
brown oil (6 g). Storage in the freezer gave a soft yellow solid
which was purified by chromatography (100 g of silica gel, 1:1
ethyl acetate/hexane) to give 3.5 g (59%) of the alcohol as a
yellow solid. The NMR showed the solid to be a mixture of
benz[f]indene-1-methanol (95%) and benz[f]indene-3-methanol.
Several recrystallizations from ligroin (bp 88.degree.-89.degree.
C.) gave 3.0 g (50.6%) of pure, colorless benz[f]indene-1-methanol:
mp 115.degree.-116.degree. C.
M. The BIMOC-Phe (1 mmole) formed in L hereinabove in dry CH.sub.2
Cl is kept under nitrogen and treated with Cyanuric fluoride (8
mmol) and pyridine (1 mmol) to form the corresponding
BIMOC-Phe-F.
Example 21
Benz [e]indene-3-methyloxycarbonylphenylalanine acid fluoride
A. Benz [e]-indene-3-methanol
1-Keto-4,5-benzindane-3-carboxylic acid, [which was prepared in
accordance with the procedure described by T. N. Poltabiraman and
W. B. Lawson in J.Biol. Chem, 247, 302a (1972) the contents of
which are incorporated herein by reference] is reduced by sodium
borohydride to form the corresponding 3-carboxylic acid -1-ol.
Dehydration with sulfuric acid give the corresponding unsaturated
acid. The unsaturated acid was then reduced by LiAlH4 to form the
3-methanol derivative: ##STR41## B.
N-[Benz[e]indene-3-methyloxycarbony]phenylalanine
This product is prepared from the product of A hereinabove by
following the procedure described in Ex. 20 I-L hereinabove
C. N-[Benz[e] indene 1-methoxycarbonyl] phenylalanine acid
fluoride
The product B formed hereinabove (1 mmol) in dry Ch.sub.2 Cl.sub.2
is kept under nitrogen and treated with cyanuric fluoride (8 mmol)
and pyridine (1 mmol) to form the above product.
Example 22
Benz [e]indene-1-methyloxy carbonylphenylalanine acid fluoride
A. Benz [e]-indene-1-methanol
3-Keto-4,5-benzindan-1-carboxylic acid, which was prepared in
accordance with the procedure described by T. N. Paltabiraman and
W. B. Lawson, in J. Biol. Chem. 242, 3029(1941) is reduced to the
corresponding 1-carboxylic acid-3-ol- by NaBH.sub.4. Dehydration
with H.sub.2 SO.sub.4 gave the unsaturated acid which was then
reduced to benz (e)-indene-1-methanol by means of LiAlH.sub.4.
##STR42## B. N-(Benz[e]indene-1-methyloxycarbonyl)
phenylalanine
This product is prepared from the product of A hereinabove by
following the procedure described in Ex. 20 I-L hereinabove
C. N-[Benz(e) indene-1-methoxyoxy carbonyl] phenylalanine acid
fluoride
The product B formed hereinabove (1 mmol) in dry CH.sub.2 Cl.sub.2
is kept under nitrogen and treated with cyanuric fluoride (8 mmol)
and pyridine (1 mmol) to form the above product.
Example 23
N-[Benz(e) indene 1-methoxy carbonyl]phenylalanine acid fluoride
and N[Benz(e) indene 3-methoxy carbonyl]phenylalanine acid
fluoride
2(.beta.)-naphthylpropionic acid is treated with thionyl chloride
and cyclized in the presence of aluminum chloride to form the
corresponding ketone. Reduction of the ketone with sodium
borohydride, followed by dehydration with sulfuric acid gives the
benz[e] indene. Formylation with ethyl formate and sodium hydride
followed by treatment with sodium borohydride gave a mixture of the
Benz[e] indene-3-methanol and Benz[e]-indene-1-methanol. The two
alcohols are separated by chromatography.
Following the procedures in Examples 22 B-C and 23 B-C, the
above-identified products are prepared.
It is to be noted from the preparations hereinabove that
Benz[e]indene 1-methoxycarbonyl and the benz[e] indene-3-methoxy
carbonyl can be used to protect the N.alpha.-amino group of an
amino acid.
These groups are formed from the corresponding alcohol, as
described in Ex. 21-23. The alcohols are then used as starting
materials to form the corresponding chloroformates and
azidoformates, as described in Ex. 20. The azidoformate can then be
reacted with an amino acid in accordance with the procedure
described in Ex. 20-L to form the corresponding N.alpha.-protected
amino acid. This then is treated with cyanuric fluoride to form the
corresponding amino acid fluoride.
Example 24
TFFH
(a) Synthesis of Tetramethylchloroformamidinum hexafluorophosphate
(TCFH). A 20% solution of phosgene in toluene (100 mL) was added
dropwise and under dry conditions to a solution of tetramethylurea
(11.6 g) in toluene. After approximately 15 min, when the carbon
dioxide evolution had stopped, anhydrous ether (350 ml) was added
under vigorous stirring. The precipitated salt was filtered and
washed with anhydrous ether (3.times.50 mL). The highly hygroscopic
material was immediately dissolved in dichloromethane (500 mL) and
to this solution a saturated solution of potassium
hexafluorophosphate (30 g/30 mL) was added under continuous
stirring for 10-15 min. The organic phase was washed with water (40
mL), dried (MgSO.sub.4), and the solvent removed under reduced
pressure to give 24.8 g (88.8%) of the salt as a white solid, mp
90.degree.-92.degree. C.; .sup.1 H NMR (CDCl.sub.3, DMSO-d.sub.6):
.delta. 3.3 (s, CH.sub.3). The same compound was prepared by using
oxalyl chloride instead of phosgene but in this case the reaction
mixture was refluxed for 2 hrs. A yield of 85.6% was obtained.
(b) Synthesis of Tetramethylfluoroformamidinium Hexafluorophosphate
(TFFH). To a solution of TCFH (5.6 g) dissolved in dry acetonitrile
(30 mL) there was added 20 mmol of KF (1.16 g, dried in the oven
for one night) portionwise and the reaction mixture was stirred at
room temperature for 2-3 hrs. The insoluble solid (KCl) was
filtered and the filtrate was evaporated with a rotary evaporator
and the residue recrystallized from acetonitrile-ether to give 4.3
g (92.3%) of the salt as white crystals, mp 111.degree.-112.degree.
C.; .sup.1 H NMR (CDCl.sub.3, DMSO-d.sub.6); .delta. 3.17-3.18 (d,
CH.sub.3). Anal. Calcd for C.sub.5 H.sub.12 F.sub.7 N.sub.2 P (mol.
wt. 264); C, 22.72; H, 4.55; N, 10.61. Found: C, 22.73; H, 4.50; N,
10.63.
Example 25
LARGE SCALE SYNTHESIS OF TFFH
The method described above was used except that 120 mL of
tetramethylurea, 200 mL of phosgene and 1000 mL of toluene were
used with 2-h stirring for the first step. After filtering and
washing with anhydrous ether the white salt was dissolved in 1500
mL of CH.sub.2 Cl.sub.2 and the solution stirred vigorously during
the addition of a saturated solution of 180 g of KPF.sub.6 in 200
mL of water (10-15 min). Then 100 mL of water was added, the
mixture shaken well in a separatory funnel and the CH.sub.2
Cl.sub.2 layer collected and dried. Removal of solvent gave 224.6 g
(80.1%) of the chloro salt. To 140.3 g (0.5 mmol) of the chloro
salt dissolved in 300 mL of CH.sub.3 CN was added with vigorous
stirring 29 g (0.5 mmol) of KF. .sup.1 H-NMR was used to follow the
reaction as the chloro derivative (.delta. 3.5 s) was converted to
the fluoro compound (.delta. 3.3, 3.29 d). After 2-3 h reaction was
complete. Filtration of KCl, evaporation and recrystallization from
CH.sub.3 CN/Et.sub.2 O gave 108 g (81.8%) of fluoro salt, mp
108.degree.-109.degree. C.
Example 26
Trimethylphenylfluoroformamidinium Hexafluorophosphate (TPFFH)
N,N-Dimethylcarbamyl chloride (21.4 g, 0.2 mol) was added to a
solution of 43.3 (0.4 mol) of N-methylaniline in 200 mL of CH.sub.2
Cl.sub.2 and the solution stirred at room temperature for 5 h. The
reaction was followed by TLC (EtOAc/hexane) and after 8 h, 200 mL
of CH.sub.2 Cl.sub.2 was added followed by 150 mL of 20%
hydrochloric acid. The mixture was stirred for 1 h, the CH.sub.2
Cl.sub.2 layer was collected, washed twice with water (200 mL),
dried and evaporated to give a colored liquid which was distilled
twice to give 21.4 g (60.1%) of the urea, bp
130.degree.-140.degree. C. (1.5-2 mm), .sup.1 H NMR (CDCl.sub.3)
.delta. 2.75 (s, 6, CH.sub.3), 3.2 (s, 3, CH.sub.3), 7-7.6 (m, 5
aryl). To 32.3 g of the urea obtained as described in 100 mL of
toluene cooled to -30.degree. to -10.degree. C. there was added 300
mL of a 20% solution of phosgene in toluene. The mixture was
stirred at this temperature for 1 h and the temperature then
allowed to come to room temperature after which the mixture was
stirred overnight, filtered, the solid washed with ether and
dissolved in 500 mL of CH.sub.2 Cl.sub.2. The solution was stirred
vigorously while 90 g of KPF.sub.6 in 120 mL of water was added
over 15 min. Water (100 mL) was added and the mixture shaken well
in a separatory funnel. The organic layer was collected, dried and
evaporated to give 51.9 g (84.0%) of the chloro salt (TPCFH) as a
white solid, mp 120.degree.-125.degree. C. (changing to a
red-colored material at 180.degree. C.), .sup.1 H-NMR (CD.sub.3
COCD.sub.3) .delta. 3.4 (s, 6, CH.sub.3), 3.8 (s, 3, CH.sub.3), 7.6
(s, 5, aryl). To 30 g (87.6 mmol) of TPCFH dissolved in 60 mL of
dry CH.sub.3 CN was added with vigorous stirring 5.2 g of KF (90
mmol). The mixture was stirred at room temperature for 2-3 h, the
reaction being followed by .sup.1 H-NMR spectroscopy. The mixture
was filtered and evaporation of the filtrate gave 25.5 g (89.3%) of
the fluoro salt after recrystallization from CH.sub.3 CN/Et.sub.2 O
as a white solid, mp 83.degree.-84.degree. C., .sup.1 H-NMR
(CD.sub.3 COCD.sub.3) .delta. 3-3.5 (m, 6, CH.sub.3), 3.8 (d, 3,
CH.sub.3), 7.6 (s, 5, aryl).
Example 27
Bis(tetramethylene)fluoroformamidinium hexafluorophosphate
(BTFFH)
To 0.01 mol of the chloro salt dissolved in 100 mL of dry
acetonitrile was added in one portion at rt, 0.015 mol of KF (dried
in an oven for 24 h). The reaction mixture was stirred overnight at
rt, filtered from KCl and washed with acetonitrile. The solvent was
removed in vacuo and the residual oily compound was dissolved in
acetonitrile and precipitated by ether. The solid was filtered and
washed with ether three times (100 mL each). The crude sample is
pure enough for further use. In order to obtain a sample for
elemental analysis, it was recrystallized from CH.sub.2 Cl.sub.2
/ether which gave white crystals, mp 153.degree.-155.degree. C.;
yield 85.2%; .sup.1 H NMR (CD.sub.3 CN) .delta. 2.03 (m, 4H), 3.84
(m, 4H).
Anal. Calcd for C.sub.9 H.sub.16 N.sub.2 PF.sub.7 : C, 34.17; N,
5.06; N, 5.06; N, 8.86. Found: C, 34.29, H, 5.09; N, 8.76.
Example 28
1,3-Dimethyl-2-fluoroimidazolium Hexafluorophosate (DFIH)
This compound was prepared from the corresponding chloro salt and
KF by the same method described above except that a reaction time
of 3h was used. The reaction can be followed by .sup.1 H NMR, by
watching the methylene singlet convert to a doublet.
Recrystallization from CH.sub.2 Cl.sub.2 /ether gave in 87.2%
yield, white crystals, mp 168.degree.-169.degree. C.; .sup.1 H NMR
(CD.sub.3 CN) .delta. 2.9 (s, 6H, CH.sub.3), 3.88 (d, 4H,
CH.sub.2); IR (KBr) 1716, 1633 (C=N.sup.+) cm.sup.-1.
Anal. Calcd for C.sub.5 H.sub.10 N.sub.2 PF.sub.7 : C, 22.9; H,
3.82; N, 10.69. Found: C, 22.1; H, 3.69; N, 10.39. The NMR spectrum
showed about 10% of the urea and if the analysis is calculated on
this basis the results agree with theory: C, 21.9; H, 3.60; N,
10.22.
Example 29
Tris(1-Pyrrolidino)fluorophosphonium hexaflurophosphate(PyFLOP)
To 10 mmol of Tris(1-pyrrolidino)-bromophosphonium
hexaflurophosphate (PyBrOP) dissolved in 30 mL of dry CH.sub.3 CN
was added 12 mmol of KF in one portion, and the mixture stirred at
rt overnight and filtered from the KBr. A test with AgNO.sub.3 gave
a heavy yellow precipitate. Acetonitrile was removed in vacuo and
the residue was recrystallized from CH.sub.2 Cl.sub.2 /ether to
give white crystals, mp 116.degree.-118.degree. C.; yield 7.9%;
.sup.1 H NMR (CD.sub.3 CN) .delta. 1.8-2.1 (m, 12H, CH.sub.2),
3.1-3.5 (M, 12H, CH.sub.2).
Anal. Calcd for C.sub.12 H.sub.24 N.sub.3 P.sub.2 F.sub.7 : C,
35.56; H, 5.931 N; 10.37. Found C, 35.28; H, 5.91; N, 10.16.
Example 30
Synthesis of Acid Fluorides Using Fluoroformamidinium Salts
General Method
To 1 mmol of protected amino acid dissolved in 5 mL of CH.sub.2
Cl.sub.2 (dry), 1 mmol of DIEA (diisopropyl ethylamine) was added,
followed by addition of 1.5 mmol of the fluoro formamidinium salt
in 5-10 mL of dry CH.sub.2 Cl.sub.2 under N.sub.2. The reaction
mixture was stirred at rt for about 3 h. IR examination showed
absorption at 1842 cm.sup.-1 after 3-5 min indicative of the COF
group, but TLC analysis showed starting material even after 1 h.
After complete reaction CH.sub.2 Cl.sub.2 can be added and the
reaction mixture washed three times with crushed ice-water (10 mL),
and the solution dried. The solvent was removed and the residue
recrystallized from CH.sub.2 Cl.sub.2 /hexane.
The following examples were prepared using the above
methodology.
__________________________________________________________________________
Synthesis of Acid Fluorides Fluoro salt Acid Fluorides mp Yield (%)
__________________________________________________________________________
##STR43## Z-Phe-F Z-Ala-F Fmoc-Phe-F 81-83.degree. C.
32-6.degree.C. (from hexane) 111-113.degree. C. 55.6 54.8 60.2
##STR44## Z-Phg- Z-Phe-F Fmoc-Tyr(tBu)-F 72-75.degree. C.
110-113.degree.C. 96-98.degree. . 60.7 63.2 63.4 ##STR45## Z-Phg-F
73-75.degree. C. 68.9
__________________________________________________________________________
Example 31
Preparation of Acid Fluorides by Use of TFFH. General Method: One
mmole of a protected amino acid and 1 mmole of pyridine were
dissolved in 10 mL of dry methylene chloride and 1.5 mmole of TFFH
was added under nitrogen. The reaction mixture was stirred at room
temperature for 3 hrs after which ice-water was added and the
organic layer separated and washed with additional ice-cold water
(2.times.10 mL), dried (MgSO.sub.4), and the solvent removed to
give an oil which was recrystallized from methylene
chloride-hexane.
Using this procedure, the following amino acid fluorides were
prepared: ##STR46##
Example 32
Examples of Racemization Tests
General Method for Coupling
To 0.25 mmol of protected amino acid dissolved in 1 mL of solvent,
0.25 mmol of DIEA was added at 0.degree. C. followed by 0.27 mmol
of TFFH or another fluoroformamidinium salt and the mixture stirred
at 0.degree. C. for 5-10 min. There was then added 0.25 mmol of an
ester.HCl or an amide and an equivalent amount of base (estere.HCl,
2 eq of base; amide, 1 eq of base). The reaction mixture was
stirred at 0.degree. C. for 1 h and at rt for 1 h. The mixture was
diluted with EtOAc (15 mL) and washed with HCl (1M), NaHCO.sub.3
(1M), saturated NaCl, and dried. Removal of solvent gave the
peptide which was checked directly by HPLC or 200 MHz .sup.1 H NMR
for racemization. The results are given below for each peptide.
TABLE I ______________________________________ Preparation of
Z--Phg--Val--OMe.sup.a CR.sup.b Solvent Base Yield (%) DL %
______________________________________ TFFH CH.sub.2 Cl.sub.2 DIEA
80.0 1.1 CH.sub.2 Cl.sub.2 TMP 75.6 <1 DMF DIEA 83.1 3.6 DMF TMP
71.2 1.6 BTFFH CH.sub.2 Cl.sub.2 DIEA 82.4 1.1 DMF DIEA 82.9 3.4
DMF TMP 70.2 1.4 DFIH CH.sub.2 Cl.sub.2 DIEA 69.9 1.3 DMF DIEA 56.9
3.5 ______________________________________ .sup.a Mp
138-140.degree. C. .sup.b Coupling reagent.
TABLE II ______________________________________ Preparation of
Z-Phg-Pro-NH.sub.2.sup.a CR Solvent Base Yield (%) DL %
______________________________________ TFFH DMF DIEA 78.9 10.17 DMF
TMP 59.8 6.9 CH.sub.2 Cl.sub.2 DIEA 76.9 4.9 CH.sub.2 Cl.sub.2 TMP
53.8 0.32 CH.sub.2 Cl.sub.2 DIEA/TMP 86.3 <0.1 BTFFH CH.sub.2
Cl.sub.2 DIEA 78.6 4.3 CH.sub.2 Cl.sub.2 DIEA/TMP 82.4 <0.1 TCFH
DMF DIEA 67.8 12.9 DMF TMP 45.4 5.43 CH.sub.2 Cl.sub.2 DIEA 78.7
6.1 CH.sub.2 Cl.sub.2 TMP 40.5 3.3 TBFH DMF DIEA 40 8.39 DMF TMP
31.7 4.7 CH.sub.2 Cl.sub.2 DIEA 45 15.9 CH.sub.2 Cl.sub.2 TMP 38.1
20.7 ##STR47## DMF DMF DIEA TMP 69.8 41.2 11.4 6.2
______________________________________ .sup.a Mp 89-92.degree. C.;
[a].sub.D.sup.23 = +54.5 (c = 1, EtOAc).
In order to establish that no racemization occurred during the
preparation of Z-Phg-F via the fluoroformamidinium salts, a sample
of the fluoride was coupled to proline amide in CH.sub.2 Cl.sub.2
in the presence of collidine. HPLC analysis showed that less than
0.1% of the DL-isomer was formed, thus establishing that no
racemization occurs during acid fluoride preparation.
TABLE III ______________________________________ Preparation of
Fmoc--His--Pro--NH.sub.2 from Fmoc--His(Trt)--OH Halo salt Base
Solvent Yield (%) DL % ______________________________________ TFFH
DIEA DMF 78.21 8.16 TMP DMF 71.8 6.4 TCFH DIEA DMF 67.8 29.2 TMP
DMF 45.4 25.5 TBFH DIEA DMF 40 51.75 TMP DMF 31.7 31.33 BTFFH DIEA
DMF 79.1 8.23 TMP DMF 73.1 6.27 DFIH DIEA DMF 76.1 8.98 TMP DMF
69.0 6.31 TFFH DIEA CH.sub.2 Cl.sub.2 80.1 4.9 TMP CH.sub.2
Cl.sub.2 69.8 1.06 DIEA/TMP (1:1) CH.sub.2 Cl.sub.2 83.4 0.46 TCFH
DIEA CH.sub.2 Cl.sub.2 78.7 6.05 TMP CH.sub.2 Cl.sub.2 60.8 3.3
TBFH DIEA CH.sub.2 Cl.sub.2 45.9 20.7 TMP CH.sub.2 Cl.sub.2 38.1
15.9 ______________________________________
TABLE IV ______________________________________ Preparation of
Fmoc--His--Pro--NH.sub.2 from Fmoc--His(Bum)--OH Salt Base Solvent
Yield (%) DL % ______________________________________ TFFH DIEA DMF
81.2 6.4 TMP DMF 68.2 1.2 TCFH DIEA DMF 71.8 15.6 TMP DMF 56.8 11.2
TBFH DIEA DMF 46.0 21.3 TMP DMF 45.0 16.5 BTFFH DIEA DMF 83.2 6.9
TMP DMF 68.7 1.3 DFIH DIEA DMF 76.2 6.7 TMP DMF 60.2 1.6
______________________________________
In the tables hereinabove and hereinbelow, TCFH is
tetramethylchoroformamidium hexaflurophosphate and TBFH is
tetramethylbromoformamidinium hexafluorophosphate, TMP is 2, 4,
6-trimethylpyridine(collidine) DIEA is disopropyl ethylamine, NMM
is N-methylmorpholine, Ps is proton sponge
(1,8-bis(dimethylamino)napthalene), DCM is methylene chloride and
ACN is acetonitrile.
From these results it is clear that coupling of trityl-protected
histidine is difficult. Best results are obtained in CH.sub.2
Cl.sub.2 using a 1:1 mixture of DIEA and collidine, the former for
activation, the latter for coupling. In addition, the
fluoroformamidinium salts are safer than the chloro and bromo
analogs. Finally, BUM protection appears to be superior to trityl
protection.
Example 33
Synthesis of FMOC-Val-F from TPFFH
A solution of 0.5 mmol of FMOC-Val-OH and 0.5 mmol of DIEA in 5 mL
of CH.sub.2 Cl.sub.2 under N.sub.2 was treated with 0.6 mmol of
TPFFH at room temperature and the reaction mixture was stirred for
2 h after which 10 mL of CH.sub.2 Cl.sub.2 was added. The solution
was washed with crushed ice, dried (MgSO.sub.4) and the solvent
removed to give a white solid which after recrystallization from
CH.sub.2 Cl.sub.2 /hexane gave the acid fluoride as white crystals,
mp 109.degree.-111.degree. C., IR (KBr) 1842 cm.sup.-1, in 68.7%
yield.
Example 34
Use of TFFH in Peptide Coupling Reactions
(1) Coupling of Z-Phe-Val-OH with H-Ala-OMe. To a solution of 0.25
mmol of Z-Phe-Val-OH (0.0995 g, 0.25 mmol), H-Ala-OMe.HCl (0.0487
g, 0.25 mmol) and 0.75 mmol of the chosen base in 1 mL of solvent
(CH.sub.2 Cl.sub.2) or DMF), cooled in an ice bath, there was added
0.3 mmol of TFFH (0.079 g), the reaction being followed by TLC
using EtOAc/hexane (7:3). Complete reaction required 4-5 hrs. After
completion of the reaction in the case of DMF the mixture was
diluted with ethyl acetate and washed with 2N HCl, 1M NaHCO.sub.3
and saturated NaCl, dried (MgSO.sub.4), the solvent removed with a
rotary evaporator and hexane added to give a white solid, mp
196.degree.-199.degree. C. which was examined in the crude state by
.sup.1 H NMR analysis at 200 MHz or HPLC analysis (Table V). As
noted the appropriate base and a lowered temperature is required
for avoidance of racemization.
TABLE V ______________________________________ Racemization During
Formation of Z--Phe--Val--Ala--OMe. DL DL DL mp of (%).sup.a
(%).sup.b (%).sup.a in crude yield in DMF in DMF CH.sub.2 Cl.sub.2
Run Base product (%) (HPLC) (.sup.1 H NMR) (HPLC)
______________________________________ 1 DIEA 195-199 72.3 24.76
25.6 2.7 2 NMM 196-199 74.5 20.85 21.9 -- 3 TMP 198-201 67.4 5.8
6.2 -- 4 PS (at RT) 194-198 75.6 7.7 8.2 -- 5 PS (at -30.degree.
C.) 196-199 56.7 1.2 <1 --
______________________________________ .sup.a Solvent system 40%
CH.sub.3 CN/60% H.sub.2 O/0.1% TFA, f = 1, .lambda..sub.214,
R.sub.t 16.3 min (LLL), 18.6 min (LDL). .sup.b Methoxy peaks:
LLL.delta. 3.75, LDL.delta. 3.70.
Example 35
(2) Coupling of Z-Phg-OH with H-Val-OMe
To 0.25 mmol of Z-Phg-OH, 0.25 mmol H-Val-OMe.HCl and 0.75 mmol of
the chosen base in 1-2 mL solvent (DMF or CH.sub.2 Cl.sub.2) at ice
bath temperature was added 0.37 mmol of TFFH. After completion of
the reaction, in the case of DMF, the reaction mixture was diluted
with ethyl acetate and washed with 1N HCl, 1M NaHCO.sub.3,
saturated NaCl, dried (MgSO.sub.4), and after removal of solvent
with a rotary evaporator the crude product was checked by .sup.1 H
NMR analysis at 200 MHz (Table VI).
TABLE VI ______________________________________ RACEMIZATION DURING
FORMATION OF Z--Phg--Val--OMe mp .degree.C. of Run Base Solvent
yield (%) crude product DL (%).sup.a
______________________________________ 1 DIEA DCM 82 137-139 1.3 2
DIEA DMF 80 136-138 3.6 3 NMM DMF 81 136-138 2.8 4 TMP DMF 82-3
136-138 1.62 ______________________________________ .sup.a Methoxy
peaks: LL.delta. 3.63, DL 3.72.
Example 36
Preparation of Z-Phg-Val-OMe via Two-Phase Coupling
To 0.5 mmol of Z-Phg-OH and 0.5 mmol of H-Val-OMe.HCl dissolved in
a mixture of 10 mL of CH.sub.2 Cl.sub.2 and 10 mL of water
containing 3 mmol of Na.sub.2 CO.sub.3 there was added with
stirring at room temperature 0.75 mmol of TFFH. After half an hour
TLC (70% EtOAc, 30% hexane) showed that only a trace of starting
material remained. After one hour an excess of CH.sub.2 Cl.sub.2
was added and the solution washed with H.sub.2 O and NaCl and dried
over MgSO.sub.4. Removal of solvent gave the dipeptide in 70.4%
yield as a white solid, mp 140.degree.-141.degree. C.; .sup.1 H NMR
(CDCl.sub.3) .delta.0.9-1.01 dd, 6H, 2CH.sub.3), 2.1-2.3 (m, 1H,
CH), 3.63 (s, 3H, OCH.sub.3), 4.5 (m, 1H, CH), 5.1 (d, 2H, CH.sub.2
O, 5.3 (d, 1H, CH), 6.1-6.2 (d, 1H, NH), 7.2-7.5 (m, 5H, aryl).
Examination of the --OMe region of the NMR spectrum (the
DL-diastereomer has its methoxy peak at .delta.3.73) showed that
only about 1.1% of the DL-isomer was present.
Example 37
Preparation of FMOC-Phe-Ala-OMe Via Two-Phase Coupling
To 0.6 mmol of FMOC-Phe-F (prepared from TFFH) in 5 mL of CH.sub.2
Cl.sub.2 there was added a mixture of H-Ala-OMe.HCl (0.5 mmol) and
Na.sub.2 CO.sub.3 (1.5 mmol) in 10 mL of CH.sub.2 Cl.sub.2 and 5 mL
of H.sub.2 O. The reaction mixture was stirred at room temperature
for 30 min and then washed with 10% KHSO.sub.4, 10% NaHCO.sub.3,
and NaCl, dried (MgSO.sub.4), and the solvent removed with a rotary
evaporator to give the dipeptide in 87.3% yield as a white solid,
mp 166.degree.-168.degree. C., .sup.1 H NMR (CDCl.sub.3) .delta.
1.3 (d, 3H, CH.sub.3), 3.1 (m, 2H, CH.sub.2), 3.73 (s, 3H,
OCH.sub.3), 4.3-4.5 (m, 5H; CH.sub.2, 5CH), 5.5 (d, 1H, NH), 6.4
(d, 1H, NH), 7.1-7.9 (m, 13H, aryl). Examination of the NMR
spectrum in the C--Me doublet region of the alanine unit (LL-
.delta.1.3 d; DL-.delta. 1.2d) showed that less than 1% of the
DL-form was present in the crude material.
Example 38
Preparation of FMOC-Phe-Ala-OMe via Direct Coupling with TFFH
To 0.5 mmol of FMOC-Phe-OH and 0.5 mmol of Ala-OMe.HCl dissolved in
a mixture of 10 mL of CH.sub.2 Cl.sub.2 and 5 mL of Na.sub.2
CO.sub.3 containing 1.5 mmol of Na.sub.2 CO.sub.3 there was added a
solution of TFFH (0.75 mmol) in 5 mL of CH.sub.2 Cl.sub.2 and the
reaction mixture was stirred at room temperature for 1 hr. An
excess of CH.sub.2 Cl.sub.2 was added and the organic layer was
separated, washed with H.sub.2 O, NaCl and dried (MgSO.sub.4).
Removal of the solvent with a rotary evaporator gave in 87.3% yield
the dipeptide as a white solid, mp 166.degree.-168.degree. C.;
.sup.1 H NMR (CDCl.sub.3) same as described above. Examination of
the .sup.1 H NMR at 200 MHz showed that less than 1% of the DL-form
was present.
The same method was used to obtain an authentic sample of the
DL-form, mp 162.degree.-164.degree. C. For the C-Me doublets, the
following data were obtained: DL-.delta.1.25 d, 3H; LL-1.35 d. It
is thus clear that there is no significant racemization during
either the synthesis of FMOC-Phe-F from TFFH or during the in situ
use of TFFH as a coupling reagent.
Example 39
Synthesis of Leucine Enkephalin in Solution via the two-Phase
Method
To 0.5 mmol of H-Leu-O-t-Bu.HCl (0.112g) and 0.5 mmol of
FMOC-Phe-OH (0.189 g) in 10 mL of CH.sub.2 Cl.sub.2 and 5 mL of 5%
Na.sub.2 CO.sub.2 which was being stirred at room temperature there
was added 0.75 mmol of TFFH (0.198 g) in 5 mL of CH.sub.2 Cl.sub.2.
The stirring was continued for 1 hr. The organic layer was
separated and washed with H.sub.2 O and saturated NaCl, dried over
MgSO.sub.4 and the solvent was removed with a rotary evaporator to
give a white solid which was dissolved in 10 mL of CH.sub.2
Cl.sub.2. The solution was used directly for deblocking by adding 7
mL of TAEA and allowing the mixture to stand for 15 min after which
it was washed with saturated NaCl (2.times.10 mL) and phosphate
buffer of pH 5.5 (3.times.15 mL). After drying over MgSO.sub.4,
removing the CH.sub.2 Cl.sub.2 and adding fresh CH.sub.2 Cl.sub.2
(10 mL) and 5 mL of Na.sub.2 CO.sub.3 (5%), there was finally added
0.5 mmole of FMOC-Gly-OH followed by 0.75 mmole of TFFH. The
work-up described above was repeated until the protected
pentapeptide FMOC-Tyr(O-t-Bu)-Gly-Gly-Phe-Leu-O-t-Bu had been
obtained in a yield of 60.7% as a white solid (0.265 g). The
protected pentapeptide was dissolved in 10 mL of CH.sub.2 Cl.sub.2
and 7 mL of TAEA added. After stirring at room temperature for 30
min the solution was washed with saturated NaCl (2.times.10 mL) and
then with pH 5.5 phosphate buffer (3.times.15 mL), dried and the
solvent was removed. After silica gel chromatography
(EtOAc/hexane/HOAc) (7:3:0.1) the pure peptide was dissolved in 50%
TFA/CH.sub.2 Cl.sub.2 (15 mL). After stirring at room temperature
for 2 hrs solvent and TFA were removed in vacuo with a water
aspirator. Ether was added at -20.degree. C. in order to
precipitate the free peptide as a white solid (0.16 g, 48.1%). The
crude sample was injected into an HPLC system using a Delta Pak
Column (5.mu., C.sub.18, 100.ANG., 3.9.times.150 mm); .ANG.220 nm;
chart speed=0.3, f=1; gradient solvent system as follows:
______________________________________ Time (min) ACN H.sub.2 O
0.1% TFA ______________________________________ 0 10 90 20 90 10 21
10 90 ______________________________________
The leucine enkephalin showed R.sub.t 15.5 min and was 93.6% pure.
Co-injection with an authentic sample which was prepared from
FMOC-AA-OPFP esters gave a single HPLC peak.
Example 40
Synthesis of Leucine Enkephalin by Solid Phase Synthesis
(a) Automatic Solid Phase Synthesis on a Millipore 9050 Instrument.
The synthesis was executed on an FMOC-Leu-PEG-PS resin under the
following conditions: Amt: 0.6 g of starting resin; coupling time:
30 min; deblocking time: 15 min; preactivation time: 7 min.
Reagents: 5 eq of FMOC-AA-OH; 5 eq of TFFH; DMF solvent. After
release from the resin and removal of the t-Bu group by treatment
with TFA for 2 hrs at room temperature, evaporation and cooling to
-30.degree. C. followed by addition of ether gave the pentapeptide
salt as a yellowish white solid in 53% yield. HPLC analysis showed
a major peak for leucine enkephalin at R.sub.t 15.05 min (88.5%
purity).
(b) Manual Solid Phase Synthesis. The synthesis was carried out on
0.2 g of the same resin described above. Conditions: deblocking
time: 15 min; coupling time: 1 hr. Reagents: 3 eqs of FMOC-AA-OH; 4
eqs of DIEA; 4 eqs of TFFH. After normal deblocking and release
from the resin precipitation gave the peptide salt as a white solid
in 56% yield. Co-injection with an authentic sample of leucine
enkephalin (R.sub.t 15.5 min, Delta Pak Column, 5.mu.) proved the
identity of the product.
Example 41
Synthesis of Leucine Enkephalin via TPCFH
The method of the previous example was followed except that 0.2 g
of FMOC-Leu-PEG-PS resin (0.21 mmol/g) was used with 10-min
deblocking time and 20-min coupling time (preactivation 2-3 min).
The peptide had a purity of 81.1%.
Example 42
Synthesis of Leucine Enkephalin via TPFFH
The method followed that of Example 41 and gave a product of 84.1%
purity.
Example 43
Comparison of Solid Phase Reactivity in the Coupling Step for TXFH,
X.dbd.F, Cl, Br
A sample of FMOC-Leu-PEG-PS (0.18 mmol/g) was deblocked by 20%
piperidine in DMF for 15 min in a plastic syringe attached to a
vacuum manifold. After deblocking, the resin was washed with three
10-mL portions of DMF, three 10-mL portions of CH.sub.2 Cl.sub.2,
two 10-mL portions of DMF and finally treated with 5 eqs of
FMOC-Val-OH, 5 eqs of the appropriate coupling reagent TXFH
(X.dbd.F, Cl, Br) and 10 eqs of DIEA in 1 mL of DMF. The
preactivation time was 2-5 min. At intervals 5-15 mg of resin was
removed, washed, deblocked with 2 mL of 20% piperidine in DMF and
the extent of coupling determined by UV analysis at 300.5 nm.
Results are listed in Table VIII.
TABLE VIII ______________________________________ PERCENT REACTION
AT VARIOUS TIMES FOR THE COUPLING OF FMOC--Val--OH TO LEUCINE
ATTACHED TO PEG--PS Time (min) TFFH TCFH TBFH TPFFH
______________________________________ 2 76.9 65 60.5 74.5 4 90.1
66.7 65.6 84.6 6 91.2 76.7 70.1 90.1 8 94.3 81.9 -- 100 10 100 86.5
79.5 99.1 15 100 91.6 84.2 100 20 100 91 86.3 100 30 98.2 97.3 91.6
98.6 ______________________________________
These results confirm that the fluoroformamidinium reagents lead to
more rapid coupling (after 10-15 min. coupling has finished for
both fluoro reagents whereas it is only 86.5-91.6% and 79.5-84.2%
complete for the chloro and bromo analogs). This agrees with the
expectation that the latter two reagents lead to oxazolone
formation from the intermediate acid chloride and acid bromide,
respectively.
Example 46
Synthesis of Prothrombin via TFFH via Solid Phase Synthesis
(Millipore 9050)
The synthesis was carried out in the normal manner described
hereinabove using 0.6 g of FMOC-PAL.sup.1 -PEG.sup.2 -PS resin
(0.25 mmol/g) with the following protocol:
(1) 1 mmol FMOC amino acid.
(2) 1 mmol of TFFH.
(3) 2 mmols of DIEA (0.6M solution in DMF, 3 mL total volume).
(4) Coupling time: 30 min.
(5) Deprotection time: 5-7 min.
(6) Preactivation time: 5-7 min.
The crude peptide showed a purity of 95% (HPLC) and on co-injection
with an authentic sample eluted at the same retention time.
Synthesis of Alamethicin Acid via Solid Phase Synthesis (Millipore
9050). The synthesis was performed on 0.5 g of FMOC-Phe-PEG-PS
(0.19 mmol/g) as described above and gave a crude peptide showing a
purity of 90% (HPLC).
.sup.1 PAL is a peptide amide linker sold by Millipore.
.sup.2 PEG-PS is polyethylene glycol polystyrene resin.
Example 45
Synthesis of ACP (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-OH)
Using TFFH
The synthesis was carried out as described hereinabove normally on
the Millipore 9050 instrument using the following conditions:
1) Fmoc-Gly-PEG-PS (0.2 mmol eq/gm).
2) 5 eq of Fmoc-AA-OH, 5 eq of TFFH, 10 eq of DIEA, 1-2 mL DMF,
conc. -0.3M.
3) preactivation time: 5-7 min.
4) coupling time: 30 min.
5) Deblocking time: 7 min, 20% piperidine/DMF.
6) Yield: 85%; purity (HPLC): 92.75.
Example 46
Coupling Via TFFH in the Absence and Presence of an Additive
A mixture of 0.125 mmol of TFFH, 0.125 mmol of HOAt, 0.125 mmol of
base and 0.5 mL of DMF was stirred at rt for 2 min and then the
mixture was added at 0.degree. C. to 0.125 mmol of an acid, 0.125
mmol of an ester.HCl or 0.125 mmol of an amide, along with 0.32
mmol of TMP for an ester salt or 0.18 mmol of TMP for a free amide.
The reaction mixture was stirred at 0.degree. C. for 1 hour, at
room temperature for 1 hour, and then worked up as usual.
______________________________________ % LDL- % LDL- Peptide
(without additive) (with additive)
______________________________________ I. Z--Phe--Val--Ala--OMe 23
1.98.sup.a <0.1.sup.b II. Z--Gly--Phe--Val--OMe 25 8.1.sup.a
<0.1.sup.b III. Z--Phe--Val--Pro--NH.sub.2 46.2 9.3.sup.a
0.65.sup.b IV. Z--Gly--Phe--Pro--NH.sub.2 -- 5.3.sup.a 0.5.sup.b V.
Z--Phe--Val--Pro--OH 47.1 -- <0.1.sup.b
______________________________________ .sup.a DIEA used for
activation (TFFH/HOAt) and TMP for coupling. .sup.b TMP used for
both activation (TFFH/HOAt) and coupling.
These results demonstrate that HOAt represents a useful additive
for use with TFFH in those cases, especially segment condensations,
where TFFH alone is not satisfactory. For solid phase syntheses
this modification is not necessary in general although it could be
advantageous in the case of .alpha.-phenylglycine or histidine.
Other examples are indicated in the table below:
______________________________________ Solid Phase Coupling of
Fmoc--Val--OH with H--Ile--PEG--PS in DMF and Comparison with
Coupling of Preformed Fmoc--Val--F CR Base Time Extent of
Coupling.sup.a ______________________________________ TFFH DIEA 2
76.9 4 90.1 6 91.2 8 94.3 10 100 15 100 20 100 Preformed DIEA 2
70.9 acid fluoride 4 78.9 6 89.6 10 99.3 15 98.8 20 100 TFFH TMP 4
16.7 6 20 10 26.5 20 32 Preformed TMP 2 62.8 acid fluoride 4 74.9 6
84.8 10 89.2 15 98.8 20 100 TFFH PS 5 57.6 10 83.6 15 81.5 20 85.7
30 97.5 Performed PS 2 69.2 acid fluoride 6 84.9 10 91.3 15 98.7 20
100 Preformed 2,6-bis(TMS)-pyridine 2 42.3 acid fluoride 4 50.4 6
52.6 10 63.1 15 85.9 20 84.3 TFFH DIEA-TMP (1:1) 2 68.9 5 79.6 10
.about.100 15 98.39 20 -100 30 -99.8
______________________________________ .sup.a UV analysis.
From these results it is clear that collidine is less effective as
an activating agent than DIEA but a 1:1 mixture of the two bases is
nearly as effective as DIEA alone. This allows collidine, which is
less likely to promote racemization, to be used for the coupling
step. It also appears that in the presence of DIEA alone TFFH is
slightly more reactive than preformed acid fluoride. This suggests
that during the activation of TFFH by DIEA there is formed an
intermediate which not only reacts with fluoride ion to give acid
fluoride, but also reacts, and reacts more readily than the acid
fluoride, with the amino group on the resin.
The above preferred embodiments and examples are given to
illustrate the scope and spirit of the present invention. These
embodiments and examples will make apparent, to those skilled in
the art, other embodiments and examples. These other embodiments
and examples are within the contemplation of the present invention.
Therefore, the present invention should be limited only by the
appended claims.
* * * * *